RESUMEN
Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.
Asunto(s)
Histonas , Lisina , Animales , Islas de CpG/genética , Metilación de ADN , Histona Metiltransferasas/genética , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Mamíferos/genética , Oogénesis/genéticaRESUMEN
Differentiation and lineage specification are controlled by cooperation of growth factor signalling. The involvement of epigenetic regulators in lineage specification remains largely elusive. Here, we show that the histone methyltransferase Mll1 prevents intestinal progenitor cells from differentiation, whereas it is also involved in secretory lineage specification of Paneth and goblet cells. Using conditional mutagenesis in mice and intestinal organoids, we demonstrate that loss of Mll1 renders intestinal progenitor cells permissive for Wnt-driven secretory differentiation. However, Mll1-deficient crypt cells fail to segregate Paneth and goblet cell fates. Mll1 deficiency causes Paneth cell-determined crypt progenitors to exhibit goblet cell features by unleashing Mapk signalling, resulting in increased numbers of mixed Paneth/goblet cells. We show that loss of Mll1 abolishes the pro-proliferative effect of Mapk signalling in intestinal progenitor cells and promotes Mapk-induced goblet cell differentiation. Our data uncover Mll1 and its downstream targets Gata4/6 as a regulatory hub of Wnt and Mapk signalling in the control of lineage specification of intestinal secretory Paneth and goblet cells.
Asunto(s)
Sistema de Señalización de MAP Quinasas/genética , Vía de Señalización Wnt/genética , Animales , Diferenciación Celular/genética , Epigénesis Genética/genética , Epigenómica/métodos , Femenino , Células Caliciformes/citología , Células Caliciformes/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Ratones , Ratones Transgénicos , Organoides/metabolismo , Células de Paneth/citología , Células de Paneth/metabolismo , Células Madre/metabolismo , Vía de Señalización Wnt/fisiologíaRESUMEN
In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron-specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.
Asunto(s)
Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Aprendizaje/fisiología , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Núcleo Celular/metabolismo , Epigénesis Genética , Hipocampo/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Integrasas/metabolismo , Memoria/fisiología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Sitio de Iniciación de la Transcripción , Transcriptoma/genéticaRESUMEN
Epigenetic mechanisms are gatekeepers for the gene expression patterns that establish and maintain cellular identity in mammalian development, stem cells and adult homeostasis. Amongst many epigenetic marks, methylation of histone 3 lysine 4 (H3K4) is one of the most widely conserved and occupies a central position in gene expression. Mixed lineage leukemia 1 (MLL1/KMT2A) is the founding mammalian H3K4 methyltransferase. It was discovered as the causative mutation in early onset leukemia and subsequently found to be required for the establishment of definitive hematopoiesis and the maintenance of adult hematopoietic stem cells. Despite wide expression, the roles of MLL1 in non-hematopoietic tissues remain largely unexplored. To bypass hematopoietic lethality, we used bone marrow transplantation and conditional mutagenesis to discover that the most overt phenotype in adult Mll1-mutant mice is intestinal failure. MLL1 is expressed in intestinal stem cells (ISCs) and transit amplifying (TA) cells but not in the villus. Loss of MLL1 is accompanied by loss of ISCs and a differentiation bias towards the secretory lineage with increased numbers and enlargement of goblet cells. Expression profiling of sorted ISCs revealed that MLL1 is required to promote expression of several definitive intestinal transcription factors including Pitx1, Pitx2, Foxa1, Gata4, Zfp503 and Onecut2, as well as the H3K27me3 binder, Bahcc1. These results were recapitulated using conditional mutagenesis in intestinal organoids. The stem cell niche in the crypt includes ISCs in close association with Paneth cells. Loss of MLL1 from ISCs promoted transcriptional changes in Paneth cells involving metabolic and stress responses. Here we add ISCs to the MLL1 repertoire and observe that all known functions of MLL1 relate to the properties of somatic stem cells, thereby highlighting the suggestion that MLL1 is a master somatic stem cell regulator.
Asunto(s)
Células Madre Adultas/fisiología , Diferenciación Celular/genética , N-Metiltransferasa de Histona-Lisina/genética , Insuficiencia Intestinal/genética , Mucosa Intestinal/patología , Proteína de la Leucemia Mieloide-Linfoide/genética , Animales , Trasplante de Médula Ósea , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Insuficiencia Intestinal/patología , Mucosa Intestinal/citología , Yeyuno/citología , Yeyuno/patología , Ratones , Ratones Transgénicos , Mutagénesis , Mutación , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Nicho de Células MadreRESUMEN
Wnt/ß-catenin signaling is crucial for intestinal carcinogenesis and the maintenance of intestinal cancer stem cells. Here we identify the histone methyltransferase Mll1 as a regulator of Wnt-driven intestinal cancer. Mll1 is highly expressed in Lgr5+ stem cells and human colon carcinomas with increased nuclear ß-catenin. High levels of MLL1 are associated with poor survival of colon cancer patients. The genetic ablation of Mll1 in mice prevents Wnt/ß-catenin-driven adenoma formation from Lgr5+ intestinal stem cells. Ablation of Mll1 decreases the self-renewal of human colon cancer spheres and halts tumor growth of xenografts. Mll1 controls the expression of stem cell genes including the Wnt/ß-catenin target gene Lgr5. Upon the loss of Mll1, histone methylation at the stem cell promoters switches from activating H3K4 tri-methylation to repressive H3K27 tri-methylation, indicating that Mll1 sustains stem cell gene expression by antagonizing gene silencing through polycomb repressive complex 2 (PRC2)-mediated H3K27 tri-methylation. Transcriptome profiling of Wnt-mutated intestinal tumor-initiating cells reveals that Mll1 regulates Gata4/6 transcription factors, known to sustain cancer stemness and to control goblet cell differentiation. Our results demonstrate that Mll1 is an essential epigenetic regulator of Wnt/ß-catenin-induced intestinal tumorigenesis and cancer stemness.
Asunto(s)
Carcinogénesis/genética , Epigénesis Genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Células Madre Neoplásicas/metabolismo , Vía de Señalización Wnt , Animales , Carcinogénesis/patología , Diferenciación Celular , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histonas/metabolismo , Humanos , Intestinos/patología , Lisina/metabolismo , Metilación , Ratones Desnudos , Células Madre Neoplásicas/patología , Complejo Represivo Polycomb 2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia Arriba/genética , Vía de Señalización Wnt/genética , beta Catenina/metabolismoRESUMEN
Methylation of histone 3 lysine 4 (H3K4) is a major epigenetic system associated with gene expression. In mammals there are six H3K4 methyltransferases related to yeast Set1 and fly Trithorax, including two orthologs of fly Trithorax-related: MLL3 and MLL4. Exome sequencing has documented high frequencies of MLL3 and MLL4 mutations in many types of human cancer. Despite this emerging importance, the requirements of these paralogs in mammalian development have only been incompletely reported. Here, we examined the null phenotypes to establish that MLL3 is first required for lung maturation, whereas MLL4 is first required for migration of the anterior visceral endoderm that initiates gastrulation in the mouse. This collective cell migration is preceded by a columnar-to-squamous transition in visceral endoderm cells that depends on MLL4. Furthermore, Mll4 mutants display incompletely penetrant, sex-distorted, embryonic haploinsufficiency and adult heterozygous mutants show aspects of Kabuki syndrome, indicating that MLL4 action, unlike MLL3, is dosage dependent. The highly specific and discordant functions of these paralogs in mouse development argues against their action as general enhancer factors.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Anomalías Múltiples/veterinaria , Alelos , Animales , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Cara/anomalías , Cara/patología , Femenino , Genotipo , Enfermedades Hematológicas/genética , Enfermedades Hematológicas/patología , Enfermedades Hematológicas/veterinaria , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Masculino , Ratones , Ratones Noqueados , Mutagénesis , Embarazo , Insuficiencia Respiratoria/etiología , Factores de Tiempo , Enfermedades Vestibulares/genética , Enfermedades Vestibulares/patología , Enfermedades Vestibulares/veterinariaRESUMEN
The Trithorax-related Set1 H3K4 methyltransferases are conserved from yeast to human. In yeast loss of Set1 causes pleiotropic effects but is compatible with life. In contrast, both mammalian Set1 orthologs: SETD1A and SETD1B are essential for embryonic development, however they have distinct functions. SETD1A is required shortly after epiblast formation whereas SETD1B becomes indispensible during early organogenesis. In adult mice both SETD1A and SETD1B regulate hematopoiesis differently: SETD1A is required for the establishment of definitive hematopoiesis whereas SETD1B is important for the maintenance of long-term hematopoietic stem cells. Both are implicated in different diseases with accumulating evidence for the association of SETD1A variants in neurological disorders and SETD1B variants with cancer. Why the two paralogs cannot or only partially compensate for the loss of each other is part of the puzzle that we try to sort out in this review.
Asunto(s)
Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Enfermedad , Drosophila , Proteínas de Drosophila , Fertilidad , Hematopoyesis/fisiología , Células Madre Hematopoyéticas , Histonas/metabolismo , Ratones , LevadurasRESUMEN
We identified a regulatory system that acts downstream of Wnt/ß-catenin signaling in salivary gland and head and neck carcinomas. We show in a mouse tumor model of K14-Cre-induced Wnt/ß-catenin gain-of-function and Bmpr1a loss-of-function mutations that tumor-propagating cells exhibit increased Mll1 activity and genome-wide increased H3K4 tri-methylation at promoters. Null mutations of Mll1 in tumor mice and in xenotransplanted human head and neck tumors resulted in loss of self-renewal of tumor-propagating cells and in block of tumor formation but did not alter normal tissue homeostasis. CRISPR/Cas9 mutagenesis and pharmacological interference of Mll1 at sequences that inhibit essential protein-protein interactions or the SET enzyme active site also blocked the self-renewal of mouse and human tumor-propagating cells. Our work provides strong genetic evidence for a crucial role of Mll1 in solid tumors. Moreover, inhibitors targeting specific Mll1 interactions might offer additional directions for therapies to treat these aggressive tumors.
Asunto(s)
Epigénesis Genética , Neoplasias de Cabeza y Cuello/genética , Código de Histonas , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Neoplasias de las Glándulas Salivales/genética , Vía de Señalización Wnt , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Dominio Catalítico , Células Cultivadas , Neoplasias de Cabeza y Cuello/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Unión Proteica , Neoplasias de las Glándulas Salivales/metabolismo , beta Catenina/metabolismoRESUMEN
Disrupting the protein-protein interaction for molecularly targeted cancer therapeutics can be a challenging but promising strategy. Compounds that disrupt the interaction between menin, a chromatin-binding protein, and oncogenic mixed lineage leukemia fusion proteins (MLL-FPs) have shown significant promise in preclinical models of leukemia and have a high degree of selectivity for leukemia versus normal hematopoietic cells. Biochemical and structural studies demonstrate that, in addition to disrupting the menin-MLL-FP interaction, such compounds also inhibit menin-MLL1, menin-MLL2, and other menin-interacting proteins. Here, we address the degree to which disruption of menin-MLL-FP interactions or menin-MLL1/MLL2 interactions contribute to the antileukemia effect of menin inhibition. We show that Men1 deletion in MLL-AF9-transformed leukemia cells produces distinct cellular and molecular consequences compared with Mll1;Mll2 co-deletion and that compounds disrupting menin-MLL N-terminal interactions largely phenocopy menin loss. Moreover, we show that Mll1;Mll2-deficient leukemia cells exhibit enhanced sensitivity to menin interaction inhibitors, which is consistent with each regulating complementary genetic pathways. These data illustrate the heightened dependency of MLL-FPs on menin compared with wild-type MLL1/MLL2 for regulation of downstream target genes and argue that the predominant action of menin inhibitory compounds is through direct inhibition of MLL-FPs without significant contribution from MLL1/MLL2 inhibition.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/metabolismo , Leucemia Mieloide Aguda/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Ratones , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Regulatory mechanisms controlling the pool size of spleen dendritic cells (DC) remain incompletely understood. DCs are continuously replenished from hematopoietic stem cells, and FLT3-mediated signals cell-intrinsically regulate homeostatic expansion of spleen DCs. Here we show that combining FLT3 and CSF1R-deficiencies results in specific and complete abrogation of spleen DCs in vivo. Spatiotemporally controlled CSF1R depletion reveals a cell-extrinsic and non-hematopoietic mechanism for DC pool size regulation. Lack of CSF1R-mediated signals impedes the differentiation of spleen macrophages of embryonic origin, and the resulted macrophage depletion during development or in adult mice results in loss of DCs. Moreover, embryo-derived macrophages are important for the physiologic regeneration of DC after activation-induced depletion in situ. In summary, we show that the differentiation of DC and their regeneration relies on ontogenetically distinct spleen macrophages, thereby providing a novel regulatory principle that may also be important for the differentiation of other hematopoietic cell types.
Asunto(s)
Células Dendríticas/citología , Macrófagos/citología , Ratones/embriología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Animales , Diferenciación Celular , Células Dendríticas/metabolismo , Femenino , Macrófagos/metabolismo , Masculino , Ratones/metabolismo , Ratones Noqueados , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Bazo/citología , Bazo/metabolismo , Tirosina Quinasa 3 Similar a fms/genética , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.
Asunto(s)
Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Homeostasis/genética , Linfopenia/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Trombocitopenia/genética , Animales , Trasplante de Médula Ósea , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Linaje de la Célula/genética , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genes Letales , Células Madre Hematopoyéticas/citología , N-Metiltransferasa de Histona-Lisina/deficiencia , Isoenzimas/deficiencia , Isoenzimas/genética , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfopenia/metabolismo , Linfopenia/patología , Ratones , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/deficiencia , Bazo/metabolismo , Bazo/patología , Trombocitopenia/metabolismo , Trombocitopenia/patología , Irradiación Corporal TotalRESUMEN
The regenerative capacity of hematopoietic stem cells (HSCs) is limited by the accumulation of DNA damage. Conditional mutagenesis of the histone 3 lysine 4 (H3K4) methyltransferase, Setd1a, revealed that it is required for the expression of DNA damage recognition and repair pathways in HSCs. Specific deletion of Setd1a in adult long-term (LT) HSCs is compatible with adult life and has little effect on the maintenance of phenotypic LT-HSCs in the bone marrow. However, SETD1A-deficient LT-HSCs lose their transcriptional cellular identity, accompanied by loss of their proliferative capacity and stem cell function under replicative stress in situ and after transplantation. In response to inflammatory stimulation, SETD1A protects HSCs and progenitors from activation-induced attrition in vivo. The comprehensive regulation of DNA damage responses by SETD1A in HSCs is clearly distinct from the key roles played by other epigenetic regulators, including the major leukemogenic H3K4 methyltransferase MLL1, or MLL5, indicating that HSC identity and function is supported by cooperative specificities within an epigenetic framework.
Asunto(s)
Proliferación Celular , Daño del ADN , Reparación del ADN , Células Madre Hematopoyéticas/enzimología , N-Metiltransferasa de Histona-Lisina/metabolismo , Animales , N-Metiltransferasa de Histona-Lisina/genética , Ratones , Ratones Noqueados , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismoRESUMEN
Histone 3 K4 trimethylation (depositing H3K4me3 marks) is typically associated with active promoters yet paradoxically occurs at untranscribed domains. Research to delineate the mechanisms of targeting H3K4 methyltransferases is ongoing. The oocyte provides an attractive system to investigate these mechanisms, because extensive H3K4me3 acquisition occurs in nondividing cells. We developed low-input chromatin immunoprecipitation to interrogate H3K4me3, H3K27ac and H3K27me3 marks throughout oogenesis. In nongrowing oocytes, H3K4me3 was restricted to active promoters, but as oogenesis progressed, H3K4me3 accumulated in a transcription-independent manner and was targeted to intergenic regions, putative enhancers and silent H3K27me3-marked promoters. Ablation of the H3K4 methyltransferase gene Mll2 resulted in loss of transcription-independent H3K4 trimethylation but had limited effects on transcription-coupled H3K4 trimethylation or gene expression. Deletion of Dnmt3a and Dnmt3b showed that DNA methylation protects regions from acquiring H3K4me3. Our findings reveal two independent mechanisms of targeting H3K4me3 to genomic elements, with MLL2 recruited to unmethylated CpG-rich regions independently of transcription.
Asunto(s)
Metilación de ADN , N-Metiltransferasa de Histona-Lisina/química , Histonas/química , Proteína de la Leucemia Mieloide-Linfoide/química , Animales , Inmunoprecipitación de Cromatina , Islas de CpG , Femenino , Cadenas de Markov , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Análisis Multivariante , Oocitos/citología , Oogénesis , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
Kmt2a and Kmt2b are H3K4 methyltransferases of the Set1/Trithorax class. We have recently shown the importance of Kmt2b for learning and memory. Here, we report that Kmt2a is also important in memory formation. We compare the decrease in H3K4 methylation and de-regulation of gene expression in hippocampal neurons of mice with knockdown of either Kmt2a or Kmt2b. Kmt2a and Kmt2b control largely distinct genomic regions and different molecular pathways linked to neuronal plasticity. Finally, we show that the decrease in H3K4 methylation resulting from Kmt2a knockdown partially recapitulates the pattern previously reported in CK-p25 mice, a model for neurodegeneration and memory impairment. Our findings point to the distinct functions of even closely related histone-modifying enzymes and provide essential insight for the development of more efficient and specific epigenetic therapies against brain diseases.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hipocampo/enzimología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Memoria , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Neuronas/enzimología , Animales , N-Metiltransferasa de Histona-Lisina/genética , Metilación , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genéticaRESUMEN
Germ cell development involves major reprogramming of the epigenome to prime the zygote for totipotency. Histone 3 lysine 4 (H3K4) methylations are universal epigenetic marks mediated in mammals by six H3K4 methyltransferases related to fly Trithorax, including two yeast Set1 orthologs: Setd1a and Setd1b. Whereas Setd1a plays no role in oogenesis, we report that Setd1b deficiency causes female sterility in mice. Oocyte-specific Gdf9-iCre conditional knockout (Setd1bGdf9 cKO) ovaries develop through all stages; however, follicular loss accumulated with age and unfertilized metaphase II (MII) oocytes exhibited irregularities of the zona pellucida and meiotic spindle. Most Setd1bGdf9 cKO zygotes remained in the pronuclear stage and displayed polyspermy in the perivitelline space. Expression profiling of Setd1bGdf9 cKO MII oocytes revealed (1) that Setd1b promotes the expression of the major oocyte transcription factors including Obox1, 2, 5, 7, Meis2 and Sall4; and (2) twice as many mRNAs were upregulated than downregulated, suggesting that Setd1b also promotes the expression of negative regulators of oocyte development with multiple Zfp-KRAB factors implicated. Together, these findings indicate that Setd1b serves as maternal effect gene through regulation of the oocyte gene expression program.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Oogénesis/genética , Oogénesis/fisiología , Animales , Blastocisto/citología , Blastocisto/metabolismo , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Factor 9 de Diferenciación de Crecimiento/deficiencia , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , N-Metiltransferasa de Histona-Lisina/deficiencia , Masculino , Herencia Materna , Ratones , Ratones Noqueados , Ratones Transgénicos , Oocitos/citología , Oocitos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula Individual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Zona Pelúcida/metabolismo , Zona Pelúcida/patología , Cigoto/citología , Cigoto/metabolismoRESUMEN
The MLL1 histone methyltransferase gene undergoes many distinct chromosomal rearrangements to yield poor-prognosis leukemia. The remaining wild-type allele is most commonly, but not always, retained. To what extent the wild-type allele contributes to leukemogenesis is unclear. Here we show, using rigorous, independent animal models, that endogenous MLL1 is dispensable for MLL-rearranged leukemia. Potential redundancy was addressed by co-deleting the closest paralog, Mll2. Surprisingly, Mll2 deletion alone had a significant impact on survival of MLL-AF9-transformed cells, and additional Mll1 loss further reduced viability and proliferation. We show that MLL1/MLL2 collaboration is not through redundancy, but regulation of distinct pathways. These findings highlight the relevance of MLL2 as a drug target in MLL-rearranged leukemia and suggest its broader significance in AML.
Asunto(s)
Proteínas de Unión al ADN/genética , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genética , Animales , Proliferación Celular/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/fisiología , Humanos , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/fisiología , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiología , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Designer nucleases like CRISPR/Cas9 enable fluent site-directed damage or small mutations in many genomes. Strategies for their use to achieve more complex tasks like regional exchanges for gene humanization or the establishment of conditional alleles are still emerging. To optimize Cas9-assisted targeting, we measured the relationship between targeting frequency and homology length in targeting constructs using a hypoxanthine-guanine phosphoribosyl-transferase assay in mouse embryonic stem cells. Targeting frequency with supercoiled plasmids improved steeply up to 2 kb total homology and continued to increase with even longer homology arms, thereby implying that Cas9-assisted targeting efficiencies can be improved using homology arms of 1 kb or greater. To humanize the Kmt2d gene, we built a hybrid mouse/human targeting construct in a bacterial artificial chromosome by recombineering. To simplify the possible outcomes, we employed a single Cas9 cleavage strategy and best achieved the intended 42 kb regional exchange with a targeting construct including a very long homology arm to recombine â¼42 kb away from the cleavage site. We recommend the use of long homology arm targeting constructs for accurate and efficient complex genome engineering, particularly when combined with the simplifying advantages of using just one Cas9 cleavage at the genome target site.
Asunto(s)
Sistemas CRISPR-Cas , Ingeniería Genética/métodos , Animales , Cromosomas Artificiales Bacterianos/genética , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/metabolismo , Endonucleasas/metabolismo , Marcación de Gen , N-Metiltransferasa de Histona-Lisina , Humanos , Hibridación Genética , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Mutación , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Neoplasias/genéticaRESUMEN
Single strand annealing proteins (SSAPs) like Redß initiate homologous recombination by annealing complementary DNA strands. We show that C-terminally truncated Redß, whilst still able to promote annealing and nucleoprotein filament formation, is unable to mediate homologous recombination. Mutations of the C-terminal domain were evaluated using both single- and double stranded (ss and ds) substrates in recombination assays. Mutations of critical amino acids affected either dsDNA recombination or both ssDNA and dsDNA recombination indicating two separable functions, one of which is critical for dsDNA recombination and the second for recombination per se. As evaluated by co-immunoprecipitation experiments, the dsDNA recombination function relates to the Redα-Redß protein-protein interaction, which requires not only contacts in the C-terminal domain but also a region near the N-terminus. Because the nucleoprotein filament formed with C-terminally truncated Redß has altered properties, the second C-terminal function could be due to an interaction required for functional filaments. Alternatively the second C-terminal function could indicate a requirement for a Redß-host factor interaction. These data further advance the model for Red recombination and the proposition that Redß and RAD52 SSAPs share ancestral and mechanistic roots.
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ADN Bacteriano/química , ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Recombinación Homóloga , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis.
Asunto(s)
Inestabilidad Genómica/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Transcripción Genética/genética , Animales , Línea Celular , Daño del ADN/genética , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones , Mutación , ARN Polimerasa II/metabolismo , RecQ Helicasas/metabolismoRESUMEN
The genetic disorder Kabuki syndrome (KS) is characterized by developmental delay and congenital anomalies. Dominant mutations in the chromatin regulators lysine (K)-specific methyltransferase 2D (KMT2D) (also known as MLL2) and lysine (K)-specific demethylase 6A (KDM6A) underlie the majority of cases. Although the functions of these chromatin-modifying proteins have been studied extensively, the physiological systems regulated by them are largely unknown. Using whole-exome sequencing, we identified a mutation in RAP1A that was converted to homozygosity as the result of uniparental isodisomy (UPD) in a patient with KS and a de novo, dominant mutation in RAP1B in a second individual with a KS-like phenotype. We elucidated a genetic and functional interaction between the respective KS-associated genes and their products in zebrafish models and patient cell lines. Specifically, we determined that dysfunction of known KS genes and the genes identified in this study results in aberrant MEK/ERK signaling as well as disruption of F-actin polymerization and cell intercalation. Moreover, these phenotypes could be rescued in zebrafish models by rebalancing MEK/ERK signaling via administration of small molecule inhibitors of MEK. Taken together, our studies suggest that the KS pathophysiology overlaps with the RASopathies and provide a potential direction for treatment design.