RESUMEN
Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L's functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.
Asunto(s)
Diferenciación Celular/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Elongación de la Transcripción Genética/fisiología , Proliferación Celular/efectos de los fármacos , Metilación de ADN/fisiología , Células Madre Embrionarias/metabolismo , Epigénesis Genética/genética , Epigenómica , Regulación de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/genética , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilación , Metiltransferasas/metabolismo , Células-Madre Neurales/metabolismo , Procesamiento Proteico-Postraduccional , Factores de Elongación Transcripcional/metabolismoRESUMEN
Catalytic-inactivating mutations within the Drosophila enhancer H3K4 mono-methyltransferase Trr and its mammalian homologs, MLL3/4, cause only minor changes in gene expression compared with whole-gene deletions for these COMPASS members. To identify essential histone methyltransferase-independent functions of Trr, we screened to identify a minimal Trr domain sufficient to rescue Trr-null lethality and demonstrate that this domain binds and stabilizes Utx in vivo. Using the homologous MLL3/MLL4 human sequences, we mapped a short â¼80-amino-acid UTX stabilization domain (USD) that promotes UTX stability in the absence of the rest of MLL3/4. Nuclear UTX stability is enhanced when the USD is fused with the MLL4 HMG-box. Thus, COMPASS-dependent UTX stabilization is an essential noncatalytic function of Trr/MLL3/MLL4, suggesting that stabilizing UTX could be a therapeutic strategy for cancers with MLL3/4 loss-of-function mutations.
Asunto(s)
Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Genes Letales/genética , N-Metiltransferasa de Histona-Lisina/genética , Oxidorreductasas N-Desmetilantes/genética , Animales , Eliminación de Gen , Regulación de la Expresión Génica/genética , Células HCT116 , Humanos , Dominios Proteicos , Estabilidad ProteicaRESUMEN
Set1A and Set1B, two members of the COMPASS family of methyltransferases that methylate the histone H3 lysine 4 (H3K4) residue, have been accredited as primary depositors of global H3K4 trimethylation (H3K4me3) in mammalian cells. Our previous studies in mouse embryonic stem cells (ESCs) demonstrated that deleting the enzymatic SET domain of Set1A does not perturb bulk H3K4me3, indicating possible compensatory roles played by other COMPASS methyltransferases. Here, we generated a series of ESC lines harboring compounding mutations of COMPASS methyltransferases. We find that Set1B is functionally redundant to Set1A in implementing H3K4me3 at highly expressed genes, while Mll2 deposits H3K4me3 at less transcriptionally active promoters. While Set1A-B/COMPASS is responsible for broad H3K4me3 peaks, Mll2/COMPASS establishes H3K4me3 with narrow breadth. Additionally, Mll2 helps preserve global H3K4me3 levels and peak breadth in the absence of Set1A-B activity. Our results illustrate the biological flexibility of such enzymes in regulating transcription in a context-dependent manner to maintain stem cell identity.
RESUMEN
It is well established that activation of the transcription factor signal transducer and activator of transcription 1 (STAT1) is required for the interferon-γ (IFN-γ)-mediated antiviral response. Here, we found that IFN-γ receptor stimulation also activated Unc-51-like kinase 1 (ULK1), an initiator of Beclin-1-mediated autophagy. Furthermore, the interaction between ULK1 and the mitogen-activated protein kinase kinase kinase MLK3 (mixed lineage kinase 3) was necessary for MLK3 phosphorylation and downstream activation of the kinase ERK5. This autophagy-independent activity of ULK1 promoted the transcription of key antiviral IFN-stimulated genes (ISGs) and was essential for IFN-γ-dependent antiviral effects. These findings define a previously unknown IFN-γ pathway that appears to be a key element of the antiviral response.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Interferón gamma/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Animales , Autofagia , Beclina-1/metabolismo , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Citocinas/metabolismo , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata , Ratones , Familia de Multigenes , Fosforilación , Unión Proteica , Receptores de Interferón/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transcripción Genética , Células U937 , Virosis/metabolismo , Receptor de Interferón gamma , Proteina Quinasa Quinasa Quinasa 11 Activada por MitógenoRESUMEN
The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Desmetilación , Elementos de Facilitación Genéticos , Receptor alfa de Estrógeno/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Sistemas CRISPR-Cas , Diferenciación Celular , Estudios de Cohortes , Metilación de ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Dioxigenasas , Epigénesis Genética , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/genética , Femenino , Humanos , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Experimentales/tratamiento farmacológico , Factor B de Elongación Transcripcional Positiva/metabolismo , Proteínas Represoras/metabolismo , Elongación de la Transcripción Genética/efectos de los fármacos , Factores de Elongación Transcripcional/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Drosophila , Femenino , Células HCT116 , Células HEK293 , Respuesta al Choque Térmico , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , Epigénesis Genética , Proteínas del Grupo Polycomb/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/metabolismo , Ratones Desnudos , Mutación/genética , Proteínas Nucleares/metabolismo , Dedos de Zinc PHD , Unión Proteica , Análisis de Supervivencia , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Chromatin regulators control cellular differentiation by orchestrating dynamic developmental gene expression programs, and hence, malfunctions in the regulation of chromatin state contribute to both developmental disorders and disease state. Mll4 (Kmt2d), a member of the COMPASS (COMplex of Proteins ASsociated with Set1) protein family that implements histone H3 lysine 4 monomethylation (H3K4me1) at enhancers, is essential for embryonic development and functions as a pancancer tumor suppressor. We define the roles of Mll4/COMPASS and its catalytic activity in the maintenance and exit of ground-state pluripotency in murine embryonic stem cells (ESCs). Mll4 is required for ESC to exit the naive pluripotent state; however, its intrinsic catalytic activity is dispensable for this process. The depletion of the H3K4 demethylase Lsd1 (Kdm1a) restores the ability of Mll4 null ESCs to transition from naive to primed pluripotency. Thus, we define an opposing regulatory axis, wherein Lsd1 and associated co-repressors directly repress Mll4-activated gene targets. This finding has broad reaching implications for human developmental syndromes and the treatment of tumors carrying Mll4 mutations.
Asunto(s)
Diferenciación Celular , Elementos de Facilitación Genéticos , Epigénesis Genética , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Complejos Multiproteicos/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/genética , Autorrenovación de las Células/genética , N-Metiltransferasa de Histona-Lisina/química , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Transcripción GenéticaRESUMEN
Gene expression in metazoans is regulated by RNA polymerase II (Pol II) promoter-proximal pausing and its release. Previously, we showed that Pol II-associated factor 1 (PAF1) modulates the release of paused Pol II into productive elongation. Here, we found that PAF1 occupies transcriptional enhancers and restrains hyperactivation of a subset of these enhancers. Enhancer activation as the result of PAF1 loss releases Pol II from paused promoters of nearby PAF1 target genes. Knockout of PAF1-regulated enhancers attenuates the release of paused Pol II on PAF1 target genes without major interference in the establishment of pausing at their cognate promoters. Thus, a subset of enhancers can primarily modulate gene expression by controlling the release of paused Pol II in a PAF1-dependent manner.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Nucleares/genética , Serpina E2/genética , Factores de Transcripción , Regulación hacia ArribaRESUMEN
Of the six members of the COMPASS (complex of proteins associated with Set1) family of histone H3 Lys4 (H3K4) methyltransferases identified in mammals, Set1A has been shown to be essential for early embryonic development and the maintenance of embryonic stem cell (ESC) self-renewal. Like its familial relatives, Set1A possesses a catalytic SET domain responsible for histone H3K4 methylation. Whether H3K4 methylation by Set1A/COMPASS is required for ESC maintenance and during differentiation has not yet been addressed. Here, we generated ESCs harboring the deletion of the SET domain of Set1A (Set1AΔSET); surprisingly, the Set1A SET domain is dispensable for ESC proliferation and self-renewal. The removal of the Set1A SET domain does not diminish bulk H3K4 methylation in ESCs; instead, only a subset of genomic loci exhibited reduction in H3K4me3 in Set1AΔSET cells, suggesting a role for Set1A independent of its catalytic domain in ESC self-renewal. However, Set1AΔSET ESCs are unable to undergo normal differentiation, indicating the importance of Set1A-dependent H3K4 methylation during differentiation. Our data also indicate that during differentiation, Set1A but not Mll2 functions as the H3K4 methylase on bivalent genes and is required for their expression, supporting a model for transcriptional switch between Mll2 and Set1A during the self-renewing-to-differentiation transition. Together, our study implicates a critical role for Set1A catalytic methyltransferase activity in regulating ESC differentiation but not self-renewal and suggests the existence of context-specific H3K4 methylation that regulates transcriptional outputs during ESC pluripotency.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/citología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Animales , Proliferación Celular/genética , Células Madre Embrionarias/enzimología , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica/genética , Metilación , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Dominios PR-SET/genéticaRESUMEN
Obesity has been linked with a host of metabolic and reproductive disorders including polycystic ovary syndrome (PCOS). While a clear association exists between obesity and PCOS, the exact nature of this relationship remains unexplained. The primary symptoms of PCOS include hyperandrogenism, anovulation, and polycystic ovaries. Most animal models utilize androgen treatments to induce PCOS. However, these models often fail to address the underlying causes of the disease and do not effectively reproduce key metabolic features such as hyperinsulinemia. Here, we present a novel rodent model of diet-induced obesity that recapitulates both the metabolic and reproductive phenotypes of human PCOS. Rats on a high-fat high-sugar (HFHS) diet not only demonstrated signs of metabolic impairment, but they also developed polycystic ovaries and experienced irregular estrous cycling. Though hyperandrogenism was not characteristic of HFHS animals as a group, elevated testosterone levels were predictive of high numbers of ovarian cysts. Alterations in steroidogenesis and folliculogenesis gene expression were also found via RNA sequencing of ovarian tissue. Importantly, the PCOS-like symptoms induced in these rats may share a similar etiology to PCOS in humans. Therefore, this model offers a unique opportunity to study PCOS at its genesis rather than following the development of disease symptoms.