RESUMEN
Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.
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Linfocitos T CD8-positivos/metabolismo , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Cisplatino/uso terapéutico , Femenino , Fibroblastos/metabolismo , Glutatión/metabolismo , Humanos , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones DesnudosRESUMEN
Although DNA N 6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2'-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.
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Adenina , Metilación de ADN , Tetrahymena thermophila , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Adenina/metabolismo , Adenina/análogos & derivados , Replicación del ADN , ADN Protozoario/genética , ADN Protozoario/metabolismoRESUMEN
INTRODUCTION: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. METHODS: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. RESULTS: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. CONCLUSION: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.
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Regulación Neoplásica de la Expresión Génica , Glioblastoma , Invasividad Neoplásica , ARN Largo no Codificante , ARN Largo no Codificante/genética , Humanos , Glioblastoma/genética , Glioblastoma/patología , Invasividad Neoplásica/genética , Línea Celular Tumoral , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Sistemas CRISPR-Cas , Técnicas de Silenciamiento del Gen , Movimiento Celular/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.
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Antígenos Nucleares/metabolismo , Histonas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Código de Histonas , Histonas/química , Humanos , Modelos Moleculares , Nucleosomas/química , Péptidos/química , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , XenopusRESUMEN
Buffering dosage imbalance of early- and late-replicating genes is important for dividing eukaryotic cells. Voichek et al. (2018) described critical roles of H3K4 methylation and Paf1C in this process, which was regulated by the S phase checkpoint and H3K56 acetylation.
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Replicación del ADN , Histonas/genética , Acetilación , Homeostasis , MetilaciónRESUMEN
The mixed-lineage leukemia (MLL)-AF10 fusion oncoprotein recruits DOT1L to the homeobox A (HOXA) gene cluster through its octapeptide motif leucine zipper (OM-LZ), thereby inducing and maintaining the MLL-AF10-associated leukemogenesis. However, the recognition mechanism between DOT1L and MLL-AF10 is unclear. Here, we present the crystal structures of both apo AF10OM-LZ and its complex with the coiled-coil domain of DOT1L. Disruption of the DOT1L-AF10 interface abrogates MLL-AF10-associated leukemic transformation. We further show that zinc stabilizes the DOT1L-AF10 complex and may be involved in the regulation of the HOXA gene expression. Our studies may also pave the way for the rational design of therapeutic drugs against MLL-rearranged leukemia.
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Transformación Celular Neoplásica/patología , Metiltransferasas , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide , Factores de Transcripción , Cristalización , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/genética , Humanos , Metiltransferasas/química , Metiltransferasas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Unión Proteica , Dominios Proteicos , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Zinc/químicaRESUMEN
A central aspect of aging research concerns the question of when individuality in lifespan arises1. Here we show that a transient increase in reactive oxygen species (ROS), which occurs naturally during early development in a subpopulation of synchronized Caenorhabditis elegans, sets processes in motion that increase stress resistance, improve redox homeostasis and ultimately prolong lifespan in those animals. We find that these effects are linked to the global ROS-mediated decrease in developmental histone H3K4me3 levels. Studies in HeLa cells confirmed that global H3K4me3 levels are ROS-sensitive and that depletion of H3K4me3 levels increases stress resistance in mammalian cell cultures. In vitro studies identified SET1/MLL histone methyltransferases as redox sensitive units of the H3K4-trimethylating complex of proteins (COMPASS). Our findings implicate a link between early-life events, ROS-sensitive epigenetic marks, stress resistance and lifespan.
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Longevidad , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Animales , Caenorhabditis elegans , Regulación hacia Abajo , Histonas/metabolismo , LarvaRESUMEN
PRDM16 is a transcription co-factor that plays critical roles in development of brown adipose tissue, as well as maintenance of adult hematopoietic and neural stem cells. Here we report that PRDM16 is a histone H3K4 methyltransferase on chromatin. Mutation in the N-terminal PR domain of PRDM16 abolishes the intrinsic enzymatic activity of PRDM16. We show that the methyltransferase activity of PRDM16 is required for specific suppression of MLL fusion protein-induced leukemogenesis both in vitro and in vivo. Mechanistic studies show that PRDM16 directly activates the SNAG family transcription factor Gfi1b, which in turn downregulates the HOXA gene cluster. Knockdown Gfi1b represses PRDM16-mediated tumor suppression, while Gfi1b overexpression mimics PRDM16 overexpression. In further support of the tumor suppressor function of PRDM16, silencing PRDM16 by DNA methylation is concomitant with MLL-AF9-induced leukemic transformation. Taken together, our study reveals a previously uncharacterized function of PRDM16 that depends on its PR domain activity.
RESUMEN
Although KMT2D, also known as MLL2, is known to play an essential role in development, differentiation, and tumor suppression, its role in pancreatic cancer development is not well understood. Here, we discovered a novel signaling axis mediated by KMT2D, which links TGF-ß to the activin A pathway. We found that TGF-ß upregulates a microRNA, miR-147b, which in turn leads to post-transcriptional silencing of KMT2D. Loss of KMT2D induces the expression and secretion of activin A, which activates a noncanonical p38 MAPK-mediated pathway to modulate cancer cell plasticity, promote a mesenchymal phenotype, and enhance tumor invasion and metastasis in mice. We observed a decreased KMT2D expression in human primary and metastatic pancreatic cancer. Furthermore, inhibition or knockdown of activin A reversed the protumoral role of KMT2D loss. These findings support a tumor-suppressive role of KMT2D in pancreatic cancer and identify miR-147b and activin A as novel therapeutic targets.
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MicroARNs , Neoplasias Pancreáticas , Humanos , Animales , Ratones , Plasticidad de la Célula , Línea Celular Tumoral , MicroARNs/genética , MicroARNs/metabolismo , Neoplasias Pancreáticas/patología , Factor de Crecimiento Transformador beta/metabolismo , Activinas/genética , Neoplasias PancreáticasRESUMEN
The MLL/KMT2 family enzymes are frequently mutated in human cancers and congenital diseases. They deposit the majority of histone 3 lysine 4 (H3K4) mono-, di-, or tri-methylation in mammals and are tightly associated with gene activation. Structural and biochemical studies in recent years provide in-depth understanding of how the MLL1 and homologous yeast SET1 complexes interact with the nucleosome core particle (NCP) and how their activities for H3K4 methylation are regulated by the conserved core components. Here, we will discuss the recent single molecule cryo-EM studies on the MLL1 and ySET1 complexes bound on the NCP. These studies highlight the dynamic regulation of the MLL/SET1 family lysine methyltransferases with unique features as compared with other histone lysine methyltransferases. These studies provide insights for loci-specific regulation of H3K4 methylation states in cells. The mechanistic studies on the MLL1 complex have already led to the development of the MLL1 inhibitors that show efficacy in acute leukemia and metastatic breast cancers. Future studies on the MLL/SET1 family enzymes will continue to bring to light potential therapeutic opportunities.
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Histonas , Proteína de la Leucemia Mieloide-Linfoide , Animales , Humanos , Histonas/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Lisina/metabolismo , Núcleo Celular/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Mamíferos/metabolismoRESUMEN
Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4(+) T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22(+) cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.
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Linfocitos T CD4-Positivos/inmunología , Neoplasias Colorrectales/inmunología , Interleucinas/inmunología , Metiltransferasas/inmunología , Células Madre Neoplásicas/inmunología , Factor de Transcripción STAT3/inmunología , Animales , Línea Celular Tumoral , Proliferación Celular , Quimiocina CCL20/inmunología , Quimiocina CCL20/metabolismo , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Activación Enzimática/inmunología , Células HT29 , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Humanos , Metiltransferasas/metabolismo , Ratones , Proteína Homeótica Nanog , Trasplante de Neoplasias , Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/inmunología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Receptores CCR6/inmunología , Receptores CCR6/metabolismo , Factores de Transcripción SOXB1/inmunología , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción STAT3/metabolismo , Interleucina-22RESUMEN
Nearly 50% of mouse and human genomes are composed of repetitive sequences. Transcription of these sequences is tightly controlled during development to prevent genomic instability, inappropriate gene activation and other maladaptive processes. Here, we demonstrate an integral role for H1 linker histones in silencing repetitive elements in mouse embryonic stem cells. Strong H1 depletion causes a profound de-repression of several classes of repetitive sequences, including major satellite, LINE-1, and ERV. Activation of repetitive sequence transcription is accompanied by decreased H3K9 trimethylation of repetitive sequence chromatin. H1 linker histones interact directly with Suv39h1, Suv39h2, and SETDB1, the histone methyltransferases responsible for H3K9 trimethylation of chromatin within these regions, and stimulate their activity toward chromatin in vitro. However, we also implicate chromatin compaction mediated by H1 as an additional, dominant repressive mechanism for silencing of repetitive major satellite sequences. Our findings elucidate two distinct, H1-mediated pathways for silencing heterochromatin.
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Cromatina/metabolismo , Histonas/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Animales , Epigenómica , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Cryo-EM structures of the KMT2A/MLL1 core complex bound on nucleosome core particles (NCPs) suggest unusual rotational dynamics of the MLL1 complex approaching its physiological substrate. However, the functional implication of such dynamics remains unclear. Here, we show that the MLL1 core complex also shows high rotational dynamics bound on the NCP carrying the catalytically inert histone H3 lysine 4 to methionine (K4M) mutation. There are two major binding modes of the MLL1 complex on the NCPK4M. Importantly, disruption of only one of the binding modes compromised the overall MLL1 activity in an NCP-specific manner. We propose that the MLL1 core complex probably exists in an equilibrium of poised and active binding modes. The high rotational dynamics of the MLL1 complex on the NCP is a feature that can be exploited for loci-specific regulation of H3K4 methylation in higher eukaryotes.
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N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Nucleosomas/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/ultraestructura , Histonas/metabolismo , Humanos , Metilación , Modelos Moleculares , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/ultraestructura , Unión Proteica , Conformación ProteicaRESUMEN
Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4. Using protein-observed 19F NMR, 1H-15N HSQC NMR, electrophoretic mobility shift assays (EMSA), and competitive-inhibition assays, we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated histone (KAc)-binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Nine additional bromodomains have also been characterized for interacting with dsDNA, including tandem BET bromodomains. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin-recognition mechanisms of the BRD4 protein for regulating gene expression.
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Histonas , Proteínas Nucleares , Cromatina , ADN , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/químicaRESUMEN
Epigenetic mechanisms that alter heritable gene expression and chromatin structure play an essential role in many biological processes, including liver function. Human MOF (males absent on the first) is a histone acetyltransferase that is globally downregulated in human steatohepatitis. However, the function of MOF in the liver remains unclear. Here, we report that MOF plays an essential role in adult liver. Genetic deletion of Mof by Mx1-Cre in the liver leads to acute liver injury, with increase of lipid deposition and fibrosis akin to human steatohepatitis. Surprisingly, hepatocyte-specific Mof deletion had no overt liver abnormality. Using the in vitro coculturing experiment, we show that Mof deletion-induced liver injury requires coordinated changes and reciprocal signaling between hepatocytes and Kupffer cells, which enables feedforward regulation to augment inflammation and apoptotic responses. At the molecular level, Mof deletion induced characteristic changes in metabolic gene programs, which bore noticeable similarity to the molecular signature of human steatohepatitis. Simultaneous deletion of Mof in both hepatocytes and macrophages results in enhanced expression of inflammatory genes and NO signaling in vitro. These changes, in turn, lead to apoptosis of hepatocytes and lipotoxicity. Our work highlights the importance of histone acetyltransferase MOF in maintaining metabolic liver homeostasis and sheds light on the epigenetic dysregulation in liver pathogenesis.
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Histona Acetiltransferasas/genética , Inflamación/metabolismo , Hepatopatías/genética , Hígado/lesiones , Óxido Nítrico/genética , Apoptosis/genética , Cromatina/genética , Epigénesis Genética/genética , Hígado Graso/genética , Hígado Graso/metabolismo , Hígado Graso/patología , Eliminación de Gen , Regulación de la Expresión Génica/genética , Hepatocitos/metabolismo , Hepatocitos/patología , Histona Acetiltransferasas/química , Humanos , Inflamación/genética , Inflamación/patología , Lípidos/efectos adversos , Lípidos/genética , Hígado/metabolismo , Hígado/patología , Hepatopatías/metabolismo , Hepatopatías/patología , Macrófagos/metabolismo , Macrófagos/patología , Óxido Nítrico/metabolismo , Transducción de Señal/genéticaRESUMEN
Hematopoietic stem cells (HSC) self-renew to sustain stem cell pools and differentiate to generate all types of blood cells. HSCs remain in quiescence to sustain their long-term self-renewal potential. It remains unclear whether protein quality control is required for stem cells in quiescence when RNA content, protein synthesis, and metabolic activities are profoundly reduced. Here, we report that protein quality control via endoplasmic reticulum-associated degradation (ERAD) governs the function of quiescent HSCs. The Sel1L/Hrd1 ERAD genes are enriched in the quiescent and inactive HSCs, and conditional knockout of Sel1L in hematopoietic tissues drives HSCs to hyperproliferation, which leads to complete loss of HSC self-renewal and HSC depletion. Mechanistically, ERAD deficiency via Sel1L knockout leads to activation of mammalian target of rapamycin (mTOR) signaling. Furthermore, we identify Ras homolog enriched in brain (Rheb), an activator of mTOR, as a novel protein substrate of Sel1L/Hrd1 ERAD, which accumulates upon Sel1L deletion and HSC activation. Importantly, inhibition of mTOR, or Rheb, rescues HSC defects in Sel1L knockout mice. Protein quality control via ERAD is, therefore, a critical checkpoint that governs HSC quiescence and self-renewal by Rheb-mediated restriction of mTOR activity.
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Proliferación Celular , Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Células Madre Hematopoyéticas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Retículo Endoplásmico/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Proteína Homóloga de Ras Enriquecida en el Cerebro/genética , Proteína Homóloga de Ras Enriquecida en el Cerebro/metabolismo , Serina-Treonina Quinasas TOR/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The mixed lineage leukaemia (MLL) family of proteins (including MLL1-MLL4, SET1A and SET1B) specifically methylate histone 3 Lys4, and have pivotal roles in the transcriptional regulation of genes involved in haematopoiesis and development. The methyltransferase activity of MLL1, by itself severely compromised, is stimulated by the three conserved factors WDR5, RBBP5 and ASH2L, which are shared by all MLL family complexes. However, the molecular mechanism of how these factors regulate the activity of MLL proteins still remains poorly understood. Here we show that a minimized human RBBP5-ASH2L heterodimer is the structural unit that interacts with and activates all MLL family histone methyltransferases. Our structural, biochemical and computational analyses reveal a two-step activation mechanism of MLL family proteins. These findings provide unprecedented insights into the common theme and functional plasticity in complex assembly and activity regulation of MLL family methyltransferases, and also suggest a universal regulation mechanism for most histone methyltransferases.
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Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Activación Enzimática , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Histone H2B ubiquitination plays an important role in transcription regulation. It has been shown that H2B ubiquitination is regulated by multiple upstream events associated with elongating RNA polymerase. Here we demonstrate that H2B K34 ubiquitylation by the MOF-MSL complex is part of the protein networks involved in early steps of transcription elongation. Knocking down MSL2 in the MOF-MSL complex affects not only global H2BK34ub, but also multiple cotranscriptionally regulated histone modifications. More importantly, we show that the MSL, PAF1, and RNF20/40 complexes are recruited and stabilized at active gene promoters by direct binary interactions. The stabilized complexes serve to regulate chromatin association of pTEFb through a positive feedback loop and facilitate Pol II transition during early transcription elongation. Results from our biochemical studies are underscored by genome-wide analyses that show high RNA Pol II processivity and transcription activity at MSL target genes.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Histonas/química , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Anticuerpos/inmunología , Sitios de Unión/genética , Línea Celular Tumoral , Cromatina/genética , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células HeLa , Histona Acetiltransferasas/química , Histonas/inmunología , Humanos , Regiones Promotoras Genéticas , Unión Proteica/genética , Interferencia de ARN , ARN Interferente Pequeño , Factores de Transcripción , Transcripción Genética , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Here we report a comprehensive characterization of our recently developed inhibitor MM-401 that targets the MLL1 H3K4 methyltransferase activity. MM-401 is able to specifically inhibit MLL1 activity by blocking MLL1-WDR5 interaction and thus the complex assembly. This targeting strategy does not affect other mixed-lineage leukemia (MLL) family histone methyltransferases (HMTs), revealing a unique regulatory feature for the MLL1 complex. Using MM-401 and its enantiomer control MM-NC-401, we show that inhibiting MLL1 methyltransferase activity specifically blocks proliferation of MLL cells by inducing cell-cycle arrest, apoptosis, and myeloid differentiation without general toxicity to normal bone marrow cells or non-MLL cells. More importantly, transcriptome analyses show that MM-401 induces changes in gene expression similar to those of MLL1 deletion, supporting a predominant role of MLL1 activity in regulating MLL1-dependent leukemia transcription program. We envision broad applications for MM-401 in basic and translational research.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Leucemia Bifenotípica Aguda/enzimología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Oligopéptidos/química , Oligopéptidos/fisiología , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
Histone modification patterns and their combinatorial readout have emerged as a fundamental mechanism for epigenetic regulation. Here we characterized Spindlin1 as a histone effector that senses a cis-tail histone H3 methylation pattern involving trimethyllysine 4 (H3K4me3) and asymmetric dimethylarginine 8 (H3R8me2a) marks. Spindlin1 consists of triple tudor-like Spin/Ssty repeats. Cocrystal structure determination established concurrent recognition of H3K4me3 and H3R8me2a by Spin/Ssty repeats 2 and 1, respectively. Both H3K4me3 and H3R8me2a are recognized using an "insertion cavity" recognition mode, contributing to a methylation state-specific layer of regulation. In vivo functional studies suggest that Spindlin1 activates Wnt/ß-catenin signaling downstream from protein arginine methyltransferase 2 (PRMT2) and the MLL complex, which together are capable of generating a specific H3 "K4me3-R8me2a" pattern. Mutagenesis of Spindlin1 reader pockets impairs activation of Wnt target genes. Taken together, our work connects a histone "lysine-arginine" methylation pattern readout by Spindlin1-to-Wnt signaling at the transcriptional level.