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1.
Nat Commun ; 15(1): 4883, 2024 Jun 07.
Article in English | MEDLINE | ID: mdl-38849395

ABSTRACT

The human methyltransferase and transcriptional coactivator MLL4 and its paralog MLL3 are frequently mutated in cancer. MLL4 and MLL3 monomethylate histone H3K4 and contain a set of uncharacterized PHD fingers. Here, we report a novel function of the PHD2 and PHD3 (PHD2/3) fingers of MLL4 and MLL3 that bind to ASXL2, a component of the Polycomb repressive H2AK119 deubiquitinase (PR-DUB) complex. The structure of MLL4 PHD2/3 in complex with the MLL-binding helix (MBH) of ASXL2 and mutational analyses reveal the molecular mechanism which is conserved in homologous ASXL1 and ASXL3. The native interaction of the Trithorax MLL3/4 complexes with the PR-DUB complex in vivo depends solely on MBH of ASXL1/2, coupling the two histone modifying activities. ChIP-seq analysis in embryonic stem cells demonstrates that MBH of ASXL1/2 is required for the deubiquitinase BAP1 recruitment to MLL4-bound active enhancers. Our findings suggest an ASXL1/2-dependent functional link between the MLL3/4 and PR-DUB complexes.


Subject(s)
DNA-Binding Proteins , Histone-Lysine N-Methyltransferase , Protein Binding , Repressor Proteins , Tumor Suppressor Proteins , Ubiquitin Thiolesterase , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Humans , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitin Thiolesterase/genetics , Repressor Proteins/metabolism , Repressor Proteins/genetics , Animals , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Mice , Enhancer Elements, Genetic , HEK293 Cells , PHD Zinc Fingers , Histones/metabolism
2.
Genome Biol ; 25(1): 143, 2024 May 31.
Article in English | MEDLINE | ID: mdl-38822412

ABSTRACT

BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Histone-Lysine N-Methyltransferase/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , Myeloid-Lymphoid Leukemia Protein/genetics , Pyrazoles/pharmacology , Pyrazoles/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cell Cycle/drug effects , Core Binding Factor Alpha 2 Subunit/genetics
3.
Sci Rep ; 14(1): 12728, 2024 06 03.
Article in English | MEDLINE | ID: mdl-38830934

ABSTRACT

To clarify the impact of SETD2 on macrophage function in pediatric patients with acute suppurative osteomyelitis and to elucidate the precise underlying mechanism. To gain insights into the potential functions of SETD2, a comprehensive study was conducted utilizing a co-culture model of human bone mesenchymal stem cells (hBMSCs) and bone marrow-derived macrophages (THP-1). A range of techniques were employed, including quantitative polymerase chain reaction, western blotting, ELISA, alkaline phosphatase activity assays, alizarin red S staining, luciferase reporter gene assays, and chromatin immunoprecipitation, to unravel the intricate interactions and molecular mechanisms involving SETD2 in this system. It was observed that SETD2 expression was reduced in THP-1 cells stimulated by staphylococcal protein A (SPA). Furthermore, the downregulation of SETD2 resulted in elevated M1 macrophage polarization and glycolysis, effects that were mitigated by SPA stimulation. Notably, SPA-stimulated THP-1 cells exhibited an increase in HIF-1α expression, which exhibited an inverse correlation with SETD2 levels. Moreover, it was discovered that SETD2 functioned as a catalyst for H3K36me3 and bound to the HIF-1α gene, which, in turn, regulated HIF-1α expression. Furthermore, the suppression of HIF-1α abrogated the consequences of SETD2 downregulation on glycolysis and M1 macrophage polarization. Lastly, the study demonstrated that M1 macrophage polarization serves as a mediator for BMP4's inhibitory effect on osteogenic differentiation of hBMSCs. This research has uncovered a previously unknown role of SETD2 in macrophages during osteomyelitis, revealing its significance in the pathogenesis of this condition. These findings suggest SETD2 as a novel target for the treatment of osteomyelitis.


Subject(s)
Cell Differentiation , Histone-Lysine N-Methyltransferase , Macrophages , Mesenchymal Stem Cells , Osteogenesis , Osteomyelitis , Humans , Osteomyelitis/metabolism , Osteomyelitis/pathology , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Macrophages/metabolism , Macrophages/immunology , Mesenchymal Stem Cells/metabolism , THP-1 Cells , Coculture Techniques , Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit
4.
Commun Biol ; 7(1): 707, 2024 Jun 08.
Article in English | MEDLINE | ID: mdl-38851815

ABSTRACT

The human protein lysine methyltransferase NSD2 catalyzes dimethylation at H3K36. It has very important roles in development and disease but many mechanistic features and its full spectrum of substrate proteins are unclear. Using peptide SPOT array methylation assays, we investigate the substrate sequence specificity of NSD2 and discover strong readout of residues between G33 (-3) and P38 (+2) on H3K36. Unexpectedly, we observe that amino acid residues different from natural ones in H3K36 are preferred at some positions. Combining four preferred residues led to the development of a super-substrate which is methylated much faster by NSD2 at peptide and protein level. Molecular dynamics simulations demonstrate that this activity increase is caused by distinct hyperactive conformations of the enzyme-peptide complex. To investigate the substrate spectrum of NSD2, we conducted a proteome wide search for nuclear proteins matching the specificity profile and discovered 22 peptide substrates of NSD2. In protein methylation studies, we identify K1033 of ATRX and K819 of FANCM as NSD2 methylation sites and also demonstrate their methylation in human cells. Both these proteins have important roles in DNA repair strengthening the connection of NSD2 and H3K36 methylation to DNA repair.


Subject(s)
Histone-Lysine N-Methyltransferase , Humans , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/genetics , Substrate Specificity , Methylation , Molecular Dynamics Simulation , Repressor Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/chemistry , Histones/metabolism , Histones/chemistry , Histones/genetics , Peptides/metabolism , Peptides/chemistry
5.
J Cell Biol ; 223(8)2024 Aug 05.
Article in English | MEDLINE | ID: mdl-38709169

ABSTRACT

Histone H3 lysine36 dimethylation (H3K36me2) is generally distributed in the gene body and euchromatic intergenic regions. However, we found that H3K36me2 is enriched in pericentromeric heterochromatin in some mouse cell lines. We here revealed the mechanism of heterochromatin targeting of H3K36me2. Among several H3K36 methyltransferases, NSD2 was responsible for inducing heterochromatic H3K36me2. Depletion and overexpression analyses of NSD2-associating proteins revealed that NSD2 recruitment to heterochromatin was mediated through the imitation switch (ISWI) chromatin remodeling complexes, such as BAZ1B-SMARCA5 (WICH), which directly binds to AT-rich DNA via a BAZ1B domain-containing AT-hook-like motifs. The abundance and stoichiometry of NSD2, SMARCA5, and BAZ1B could determine the localization of H3K36me2 in different cell types. In mouse embryos, H3K36me2 heterochromatin localization was observed at the two- to four-cell stages, suggesting its physiological relevance.


Subject(s)
Chromatin Assembly and Disassembly , Heterochromatin , Histone-Lysine N-Methyltransferase , Histones , Repressor Proteins , Animals , Humans , Mice , Adenosine Triphosphatases , Bromodomain Containing Proteins/genetics , Bromodomain Containing Proteins/metabolism , Centromere/metabolism , Centromere/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Heterochromatin/metabolism , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Histones/genetics , Methylation , Repressor Proteins/metabolism , Repressor Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics
6.
Rev Neurol ; 78(10): 285-291, 2024 May 16.
Article in Spanish | MEDLINE | ID: mdl-38743022

ABSTRACT

INTRODUCTION: KMT2B-related dystonia is a childhood-onset movement disorder characterized by focal dystonia of the lower extremities progressing to generalized dystonia with predominant cervical, cranial, and laryngeal involvement. So far, fewer than 100 variants have been reported, the vast majority being de novo mutations. The presenting frame of KMT2B dystonia, with dysmorphology features and other complex neurologic symptoms shows the spectrum of KMT2B dystonia as a probable syndromic disease, rather than an isolated early-onset dystonia, which has been the classic view of the condition. CASE REPORTS: We report three patients who presented regression in their neurodevelopment, focal dystonia of the lower limbs with subsequent generalization, in whom two de novo variants were reported in the KMT2B gene, with a mean age of presentation lower than the average reported worldwide. CONCLUSIONS: We describe the largest local series of patients with KMT2B dystonia in Colombia (to our knowledge), which allows us to expand the genotype-phenotype relationship of this genetic dystonia. Although many affected individuals follow a similar disease course, it is important to determine clinical features that may be associated such as neurodevelopmental regression.


TITLE: Espectro fenotípico y genotípico de la distonía causada por el gen KMT2B. Descripción de tres casos colombianos.Introducción. La distonía relacionada con KMT2B es un trastorno del movimiento hipercinético, de inicio en la infancia, caracterizado inicialmente por distonía focal de las extremidades inferiores que progresa a una distonía generalizada con mayor afectación cervical, craneal y laríngea. Hasta el momento se han descrito aproximadamente 100 variantes causantes de enfermedad, y la mayoría son de novo. La presentación clínica de la distonía con hallazgos fenotípicos comunes en los pacientes, asociada a frecuente afectación del neurodesarrollo, afectación intelectual y otros trastornos del movimiento, hace pensar más en el espectro KMT2B como una condición sindrómica, más que en una distonía aislada de inicio temprano, como clásicamente se ha clasificado. Casos clínicos. Se presenta el caso de tres pacientes con regresión del neurodesarrollo, distonía focal de los miembros inferiores con posterior generalización, en quienes se identificaron tres variantes en el gen KMT2B, en dos de los casos de novo (en una de ellas se desconoce la segregación), y dos de ellas comunicadas por primera vez en la bibliografía. La edad media de presentación fue menor a la edad promedio notificada a nivel mundial. Conclusiones. Describimos una serie de pacientes colombianos con distonía DYT-KMT2B (la más grande en nuestro conocimiento a nivel local), lo que nos permite ampliar la relación genotipo-fenotipo de esta distonía genética. Si bien múltiples individuos afectados siguen un curso de enfermedad similar, es importante determinar las características clínicas que pueden estar asociadas, como la regresión del neurodesarrollo y su potencial clasificación como distonía compleja. Describimos, además, dos nuevas variantes asociadas al fenotipo.


Subject(s)
Histone-Lysine N-Methyltransferase , Phenotype , Humans , Colombia , Male , Female , Histone-Lysine N-Methyltransferase/genetics , Child , Adolescent , Genotype , Dystonic Disorders/genetics , Mutation
7.
Nat Commun ; 15(1): 4023, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38740816

ABSTRACT

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.


Subject(s)
Cytokinesis , Endosomal Sorting Complexes Required for Transport , Endosomal Sorting Complexes Required for Transport/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Humans , Methylation , HeLa Cells , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , HIV-1/metabolism , HIV-1/genetics , HIV-1/physiology , Lysine/metabolism , Protein Processing, Post-Translational
8.
Front Immunol ; 15: 1341745, 2024.
Article in English | MEDLINE | ID: mdl-38765012

ABSTRACT

Individuals with Kabuki syndrome present with immunodeficiency; however, how pathogenic variants in the gene encoding the histone-modifying enzyme lysine methyltransferase 2D (KMT2D) lead to immune alterations remain poorly understood. Following up on our prior report of KMT2D-altered integrin expression in B-cells, we performed targeted analyses of KMT2D's influence on integrin expression in T-cells throughout development (thymocytes through peripheral T-cells) in murine cells with constitutive- and conditional-targeted Kmt2d deletion. Using high-throughput RNA-sequencing and flow cytometry, we reveal decreased expression (both at the transcriptional and translational levels) of a cluster of leukocyte-specific integrins, which perturb aspects of T-cell activation, maturation, adhesion/localization, and effector function. H3K4me3 ChIP-PCR suggests that these evolutionary similar integrins are under direct control of KMT2D. KMT2D loss also alters multiple downstream programming/signaling pathways, including integrin-based localization, which can influence T-cell populations. We further demonstrated that KMT2D deficiency is associated with the accumulation of murine CD8+ single-positive (SP) thymocytes and shifts in both human and murine peripheral T-cell populations, including the reduction of the CD4+ recent thymic emigrant (RTE) population. Together, these data show that the targeted loss of Kmt2d in the T-cell lineage recapitulates several distinct features of Kabuki syndrome-associated immune deficiency and implicates epigenetic mechanisms in the regulation of integrin signaling.


Subject(s)
Integrins , Lymphocyte Activation , Animals , Mice , Integrins/metabolism , Integrins/genetics , Lymphocyte Activation/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Mice, Knockout , Vestibular Diseases/genetics , Vestibular Diseases/immunology , Vestibular Diseases/metabolism , Face/abnormalities , Humans , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Mice, Inbred C57BL , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Neoplasm Proteins/metabolism , Signal Transduction , Gene Expression Regulation , Abnormalities, Multiple , Hematologic Diseases , Myeloid-Lymphoid Leukemia Protein
9.
Sci Adv ; 10(18): eadj8042, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38691608

ABSTRACT

Overactivation of the transforming growth factor-ß (TGFß) signaling in Duchenne muscular dystrophy (DMD) is a major hallmark of disease progression, leading to fibrosis and muscle dysfunction. Here, we investigated the role of SETDB1 (SET domain, bifurcated 1), a histone lysine methyltransferase involved in muscle differentiation. Our data show that, following TGFß induction, SETDB1 accumulates in the nuclei of healthy myotubes while being already present in the nuclei of DMD myotubes where TGFß signaling is constitutively activated. Transcriptomics revealed that depletion of SETDB1 in DMD myotubes leads to down-regulation of TGFß target genes coding for secreted factors involved in extracellular matrix remodeling and inflammation. Consequently, SETDB1 silencing in DMD myotubes abrogates the deleterious effect of their secretome on myoblast differentiation by impairing myoblast pro-fibrotic response. Our findings indicate that SETDB1 potentiates the TGFß-driven fibrotic response in DMD muscles, providing an additional axis for therapeutic intervention.


Subject(s)
Histone-Lysine N-Methyltransferase , Muscle Fibers, Skeletal , Muscular Dystrophy, Duchenne , Signal Transduction , Transforming Growth Factor beta , Muscular Dystrophy, Duchenne/metabolism , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/pathology , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Muscle Fibers, Skeletal/metabolism , Muscle Fibers, Skeletal/pathology , Transforming Growth Factor beta/metabolism , Humans , Animals , Cell Differentiation , Mice , Myoblasts/metabolism , Fibrosis , Gene Expression Regulation
10.
Biol Direct ; 19(1): 41, 2024 May 29.
Article in English | MEDLINE | ID: mdl-38812048

ABSTRACT

The enzymes performing protein post-translational modifications (PTMs) form a critical post-translational regulatory circuitry that orchestrates literally all cellular processes in the organism. In particular, the balance between cellular stemness and differentiation is crucial for the development of multicellular organisms. Importantly, the fine-tuning of this balance on the genetic level is largely mediated by specific PTMs of histones including lysine methylation. Lysine methylation is carried out by special enzymes (lysine methyltransferases) that transfer the methyl group from S-adenosyl-L-methionine to the lysine residues of protein substrates. Set7/9 is one of the exemplary protein methyltransferases that however, has not been fully studied yet. It was originally discovered as histone H3 lysine 4-specific methyltransferase, which later was shown to methylate a number of non-histone proteins that are crucial regulators of stemness and differentiation, including p53, pRb, YAP, DNMT1, SOX2, FOXO3, and others. In this review we summarize the information available to date on the role of Set7/9 in cellular differentiation and tissue development during embryogenesis and in adult organisms. Finally, we highlight and discuss the role of Set7/9 in pathological processes associated with aberrant cellular differentiation and self-renewal, including the formation of cancer stem cells.


Subject(s)
Cell Differentiation , Histone-Lysine N-Methyltransferase , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Humans , Animals , Protein Processing, Post-Translational , Methylation , Stem Cells/metabolism
11.
Proc Natl Acad Sci U S A ; 121(23): e2401973121, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38809707

ABSTRACT

In many mammals, recombination events are concentrated in hotspots directed by a sequence-specific DNA-binding protein named PRDM9. Intriguingly, PRDM9 has been lost several times in vertebrates, and notably among mammals, it has been pseudogenized in the ancestor of canids. In the absence of PRDM9, recombination hotspots tend to occur in promoter-like features such as CpG islands. It has thus been proposed that one role of PRDM9 could be to direct recombination away from PRDM9-independent hotspots. However, the ability of PRDM9 to direct recombination hotspots has been assessed in only a handful of species, and a clear picture of how much recombination occurs outside of PRDM9-directed hotspots in mammals is still lacking. In this study, we derived an estimator of past recombination activity based on signatures of GC-biased gene conversion in substitution patterns. We quantified recombination activity in PRDM9-independent hotspots in 52 species of boreoeutherian mammals. We observe a wide range of recombination rates at these loci: several species (such as mice, humans, some felids, or cetaceans) show a deficit of recombination, while a majority of mammals display a clear peak of recombination. Our results demonstrate that PRDM9-directed and PRDM9-independent hotspots can coexist in mammals and that their coexistence appears to be the rule rather than the exception. Additionally, we show that the location of PRDM9-independent hotspots is relatively more stable than that of PRDM9-directed hotspots, but that PRDM9-independent hotspots nevertheless evolve slowly in concert with DNA hypomethylation.


Subject(s)
Histone-Lysine N-Methyltransferase , Recombination, Genetic , Animals , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Mammals/genetics , CpG Islands/genetics , Eutheria/genetics , Mice , Female , Gene Conversion , Evolution, Molecular
12.
J Exp Clin Cancer Res ; 43(1): 151, 2024 May 30.
Article in English | MEDLINE | ID: mdl-38812026

ABSTRACT

BACKGROUND: SMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair. METHODS: In cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients' residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs). RESULTS: Here we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models. CONCLUSIONS: Overall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.


Subject(s)
DNA Damage , DNA Repair , Drug Resistance, Neoplasm , Histone-Lysine N-Methyltransferase , Humans , Animals , Mice , DNA Repair/drug effects , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Cell Line, Tumor , Xenograft Model Antitumor Assays , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Female
13.
Cells ; 13(10)2024 May 19.
Article in English | MEDLINE | ID: mdl-38786098

ABSTRACT

Breast cancer develops upon sequential acquisition of driver mutations in mammary epithelial cells; however, how these mutations collaborate to transform normal cells remains unclear in most cases. We aimed to reconstitute this process in a particular case. To this end, we combined the activated form of the PI 3-kinase harboring the H1047R mutation with the inactivation of the histone lysine methyl-transferase KMT2D in the non-tumorigenic human mammary epithelial cell line MCF10A. We found that PI 3-kinase activation promoted cell-cycle progression, especially when growth signals were limiting, as well as cell migration, both in a collective monolayer and as single cells. Furthermore, we showed that KMT2D inactivation had relatively little influence on these processes, except for single-cell migration, which KMT2D inactivation promoted in synergy with PI 3-kinase activation. The combination of these two genetic alterations induced expression of the ARPC5L gene that encodes a subunit of the Arp2/3 complex. ARPC5L depletion fully abolished the enhanced migration persistence exhibited by double-mutant cells. Our reconstitution approach in MCF10A has thus revealed both the cell function and the single-cell migration, and the underlying Arp2/3-dependent mechanism, which are synergistically regulated when KMT2D inactivation is combined with the activation of the PI 3-kinase.


Subject(s)
Actin-Related Protein 2-3 Complex , Cell Movement , Epithelial Cells , Histone-Lysine N-Methyltransferase , Phosphatidylinositol 3-Kinases , Humans , Cell Movement/genetics , Epithelial Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Actin-Related Protein 2-3 Complex/metabolism , Actin-Related Protein 2-3 Complex/genetics , Female , Mammary Glands, Human/metabolism , Mammary Glands, Human/cytology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Mutation/genetics , Cell Line
14.
Int J Mol Sci ; 25(9)2024 Apr 26.
Article in English | MEDLINE | ID: mdl-38731926

ABSTRACT

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene (MLL) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in ßHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2. Similar observations were made in MLL heterozygous knockout mice (MLL+/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.


Subject(s)
Glucose Transporter Type 2 , Glucose , Histone-Lysine N-Methyltransferase , Insulin Secretion , Insulin-Secreting Cells , Myeloid-Lymphoid Leukemia Protein , Animals , Insulin-Secreting Cells/metabolism , Glucose/metabolism , Mice , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Glucose Transporter Type 2/metabolism , Glucose Transporter Type 2/genetics , Gene Expression Regulation , Mice, Knockout , Insulin/metabolism , Glucose Transporter Type 1/metabolism , Glucose Transporter Type 1/genetics , Cell Line , Male
15.
Cell Rep ; 43(5): 114222, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38735046

ABSTRACT

The activation and specialization of regulatory T cells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that T cell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional T cells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization.


Subject(s)
Histone-Lysine N-Methyltransferase , Lymphocyte Activation , Myeloid-Lymphoid Leukemia Protein , T-Lymphocytes, Regulatory , Animals , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Mice , Mice, Inbred C57BL , Histones/metabolism , T-Box Domain Proteins/metabolism , T-Box Domain Proteins/genetics , Methylation , Cell Proliferation
16.
Cell Rep ; 43(5): 114174, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38700982

ABSTRACT

Activating mutations in PIK3CA are frequently found in estrogen-receptor-positive (ER+) breast cancer, and the combination of the phosphatidylinositol 3-kinase (PI3K) inhibitor alpelisib with anti-ER inhibitors is approved for therapy. We have previously demonstrated that the PI3K pathway regulates ER activity through phosphorylation of the chromatin modifier KMT2D. Here, we discovered a methylation site on KMT2D, at K1330 directly adjacent to S1331, catalyzed by the lysine methyltransferase SMYD2. SMYD2 loss attenuates alpelisib-induced KMT2D chromatin binding and alpelisib-mediated changes in gene expression, including ER-dependent transcription. Knockdown or pharmacological inhibition of SMYD2 sensitizes breast cancer cells, patient-derived organoids, and tumors to PI3K/AKT inhibition and endocrine therapy in part through KMT2D K1330 methylation. Together, our findings uncover a regulatory crosstalk between post-translational modifications that fine-tunes KMT2D function at the chromatin. This provides a rationale for the use of SMYD2 inhibitors in combination with PI3Kα/AKT inhibitors in the treatment of ER+/PIK3CA mutant breast cancer.


Subject(s)
Breast Neoplasms , Chromatin , Histone-Lysine N-Methyltransferase , Humans , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Breast Neoplasms/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Female , Chromatin/metabolism , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Methylation/drug effects , Cell Line, Tumor , Animals , Mice , Proto-Oncogene Proteins c-akt/metabolism , Neoplasm Proteins/metabolism , Neoplasm Proteins/genetics , Receptors, Estrogen/metabolism , Gene Expression Regulation, Neoplastic/drug effects
17.
Nat Commun ; 15(1): 4176, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38755176

ABSTRACT

SETD3 is an essential host factor for the replication of a variety of enteroviruses that specifically interacts with viral protease 2A. However, the interaction between SETD3 and the 2A protease has not been fully characterized. Here, we use X-ray crystallography and cryo-electron microscopy to determine the structures of SETD3 complexed with the 2A protease of EV71 to 3.5 Å and 3.1 Å resolution, respectively. We find that the 2A protease occupies the V-shaped central cleft of SETD3 through two discrete sites. The relative positions of the two proteins vary in the crystal and cryo-EM structures, showing dynamic binding. A biolayer interferometry assay shows that the EV71 2A protease outcompetes actin for SETD3 binding. We identify key 2A residues involved in SETD3 binding and demonstrate that 2A's ability to bind SETD3 correlates with EV71 production in cells. Coimmunoprecipitation experiments in EV71 infected and 2A expressing cells indicate that 2A interferes with the SETD3-actin complex, and the disruption of this complex reduces enterovirus replication. Together, these results reveal the molecular mechanism underlying the interplay between SETD3, actin, and viral 2A during virus replication.


Subject(s)
Actins , Cryoelectron Microscopy , Enterovirus A, Human , Protein Binding , Humans , Actins/metabolism , Enterovirus A, Human/genetics , Enterovirus A, Human/metabolism , Crystallography, X-Ray , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/chemistry , Virus Replication , Viral Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/chemistry , Enterovirus Infections/virology , Enterovirus Infections/metabolism , Models, Molecular , Histone Methyltransferases
18.
Protein Sci ; 33(6): e5019, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38747396

ABSTRACT

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.


Subject(s)
Peptidomimetics , Peptidomimetics/chemistry , Peptidomimetics/metabolism , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Intrinsically Disordered Proteins/genetics , Intrinsically Disordered Proteins/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Models, Molecular , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Crystallography, X-Ray , Protein Domains , Myeloid-Lymphoid Leukemia Protein/chemistry , Myeloid-Lymphoid Leukemia Protein/metabolism , Myeloid-Lymphoid Leukemia Protein/genetics , Myeloid-Lymphoid Leukemia Protein/antagonists & inhibitors
19.
Cell Death Dis ; 15(5): 335, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38744853

ABSTRACT

PTENα/ß, two variants of PTEN, play a key role in promoting tumor growth by interacting with WDR5 through their N-terminal extensions (NTEs). This interaction facilitates the recruitment of the SET1/MLL methyltransferase complex, resulting in histone H3K4 trimethylation and upregulation of oncogenes such as NOTCH3, which in turn promotes tumor growth. However, the molecular mechanism underlying this interaction has remained elusive. In this study, we determined the first crystal structure of PTENα-NTE in complex with WDR5, which reveals that PTENα utilizes a unique binding motif of a sequence SSSRRSS found in the NTE domain of PTENα/ß to specifically bind to the WIN site of WDR5. Disruption of this interaction significantly impedes cell proliferation and tumor growth, highlighting the potential of the WIN site inhibitors of WDR5 as a way of therapeutic intervention of the PTENα/ß associated cancers. These findings not only shed light on the important role of the PTENα/ß-WDR5 interaction in carcinogenesis, but also present a promising avenue for developing cancer treatments that target this pathway.


Subject(s)
Intracellular Signaling Peptides and Proteins , PTEN Phosphohydrolase , PTEN Phosphohydrolase/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/chemistry , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/chemistry , Animals , Mice , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/metabolism , Cell Proliferation/genetics , Disease Progression , Protein Binding , Cell Line, Tumor , Mice, Nude , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/chemistry , Protein Domains , Amino Acid Motifs
20.
BMC Biol ; 22(1): 105, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38702628

ABSTRACT

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.


Subject(s)
Histone-Lysine N-Methyltransferase , Histones , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Histones/metabolism , Histones/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Methylation , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/genetics
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