RESUMEN
Improvements in tumor immunotherapies depend on better understanding of the anti-tumor T cell response. By studying human tumor-draining lymph nodes (TDLNs), we found that activated CD8+ T cells in TDLNs shared functional, transcriptional, and epigenetic traits with TCF1+ stem-like cells in the tumor. The phenotype and TCR overlap suggested that these TDLN cells were precursors to tumor-resident stem-like CD8+ T cells. Murine tumor models revealed that tumor-specific CD8+ T cells were activated in TDLNs but lacked an effector phenotype. These stem-like cells migrated into the tumor, where additional co-stimulation from antigen-presenting cells drove effector differentiation. This model of CD8+ T cell activation in response to cancer is different from that of canonical CD8+ T cell activation to acute viruses, and it proposes two stages of tumor-specific CD8+ T cell activation: initial activation in TDLNs and subsequent effector program acquisition within the tumor after additional co-stimulation.
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Linfocitos T CD8-positivos , Neoplasias , Humanos , Animales , Ratones , Neoplasias/patología , Ganglios Linfáticos , Activación de Linfocitos , Diferenciación CelularRESUMEN
Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.
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Subgrupos de Linfocitos B/patología , Metilación de ADN/genética , Epigénesis Genética/genética , Lupus Eritematoso Sistémico/genética , Subgrupos de Linfocitos B/inmunología , Ensamble y Desensamble de Cromatina/fisiología , Factores de Transcripción de la Respuesta de Crecimiento Precoz/genética , Humanos , Lupus Eritematoso Sistémico/inmunología , Factor de Transcripción AP-1/genética , Transcriptoma/genéticaRESUMEN
The epigenetic processes that regulate antibody-secreting plasma cells are not well understood. Here, analysis of plasma cell differentiation revealed DNA hypomethylation of 10% of CpG loci that were overrepresented at enhancers. Inhibition of DNA methylation enhanced plasma cell commitment in a cell-division-dependent manner. Analysis of B cells differentiating in vivo stratified by cell division revealed a fivefold increase in mRNA transcription coupled to DNA hypomethylation. Demethylation occurred first at binding motifs for the transcription factors NF-κB and AP-1 and later at those for the transcription factors IRF and Oct-2 and was coincident with activation and differentiation gene-expression programs in a cell-division-dependent manner. These data provide mechanistic insight into cell-division-coupled transcriptional and epigenetic reprogramming and suggest that DNA hypomethylation reflects the cis-regulatory history of plasma cell differentiation.
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Linfocitos B/fisiología , Metilación de ADN , FN-kappa B/metabolismo , Células Plasmáticas/fisiología , Factor de Transcripción AP-1/metabolismo , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , División Celular/genética , Células Cultivadas , Islas de CpG/genética , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Inmunidad Humoral/genética , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , Factor 2 de Transcripción de Unión a Octámeros/genética , Factor 2 de Transcripción de Unión a Octámeros/metabolismo , Factor de Transcripción AP-1/genéticaRESUMEN
ABSTRACT: The translocation t(11;14) occurs in 20% of patients with multiple myeloma (MM) and results in the upregulation of CCND1. Nearly two-thirds of t(11;14) MM cells are BCL2 primed and highly responsive to the oral BCL2 inhibitor venetoclax. Although it is evident that this unique sensitivity to venetoclax depends on the Bcl-2 homology domain 3- proapoptotic protein priming of BCL2, the biology underlying t(11;14) MM dependency on BCL2 is poorly defined. Importantly, the epigenetic regulation of t(11;14) transcriptomes and its impact on gene regulation and clinical response to venetoclax remain elusive. In this study, by integrating assay for transposase-accessible chromatin by sequencing (ATAC-seq) and RNA-seq at the single-cell level in primary MM samples, we have defined the epigenetic regulome and transcriptome associated with t(11;14) MM. A B-cell-like epigenetic signature was enriched in t(11;14) MM, confirming its phylogeny link to B-cell rather than plasma cell biology. Of note, a loss of a B-cell-like epigenetic signature with a gain of canonical plasma cell transcription factors was observed at the time of resistance to venetoclax. In addition, MCL1 and BCL2L1 copy number gains and structural rearrangements were linked to venetoclax resistance in patients with t(11;14) MM. To date, this is the first study in which both single-cell (sc) ATAC-seq and scRNA-seq analysis are integrated into primary MM cells to obtain a deeper resolution of the epigenetic regulome and transcriptome associated with t(11;14) MM biology and venetoclax resistance.
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Antineoplásicos , Mieloma Múltiple , Humanos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Epigénesis Genética , Antineoplásicos/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéuticoRESUMEN
The histone H3K27 demethylase KDM6A is a tumor suppressor in multiple cancers, including multiple myeloma (MM). We created isogenic MM cells disrupted for KDM6A and tagged the endogenous protein to facilitate genome wide studies. KDM6A binds genes associated with immune recognition and cytokine signaling. Most importantly, KDM6A binds and activates NLRC5 and CIITA encoding regulators of Major Histocompatibility Complex (MHC) genes. Patient data indicate that NLRC5 and CIITA, are downregulated in MM with low KDM6A expression. Chromatin analysis shows that KDM6A binds poised and active enhancers and KDM6A loss led to decreased H3K27ac at enhancers, increased H3K27me3 levels in body of genes bound by KDM6A and decreased gene expression. Reestablishing histone acetylation with an HDAC3 inhibitor leads to upregulation of MHC expression, offering a strategy to restore immunogenicity of KDM6A deficient tumors. Loss of Kdm6a in murine RAS-transformed fibroblasts led to increased growth in vivo associated with decreased T cell infiltration.
RESUMEN
ABSTRACT: Chromosomal translocation (4;14), an adverse prognostic factor in multiple myeloma (MM), drives overexpression of the histone methyltransferase nuclear receptor binding SET domain protein 2 (NSD2). A genome-wide CRISPR screen in MM cells identified adenylate kinase 2 (AK2), an enzyme critical for high-energy phosphate transfer from the mitochondria, as an NSD2-driven vulnerability. AK2 suppression in t(4;14) MM cells decreased nicotinamide adenine dinucleotide phosphate (NADP[H]) critical for conversion of ribonucleotides to deoxyribonucleosides, leading to replication stress, DNA damage, and apoptosis. Driving a large genome-wide increase in chromatin methylation, NSD2 overexpression depletes S-adenosylmethionine, compromising the synthesis of creatine from its precursor, guanidinoacetate. Creatine supplementation restored NADP(H) levels, reduced DNA damage, and rescued AK2-deficient t(4;14) MM cells. As the creatine phosphate shuttle constitutes an alternative means for mitochondrial high-energy phosphate transport, these results indicate that NSD2-driven creatine depletion underlies the hypersensitivity of t(4;14) MM cells to AK2 loss. Furthermore, AK2 depletion in t(4;14) cells impaired protein folding in the endoplasmic reticulum, consistent with impaired use of mitochondrial adenosine triphosphate (ATP). Accordingly, AK2 suppression increased the sensitivity of MM cells to proteasome inhibition. These findings delineate a novel mechanism in which aberrant transfer of carbon to the epigenome creates a metabolic vulnerability, with direct therapeutic implications for t(4;14) MM.
Asunto(s)
Adenilato Quinasa , N-Metiltransferasa de Histona-Lisina , Mieloma Múltiple , Translocación Genética , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Adenilato Quinasa/metabolismo , Adenilato Quinasa/genética , Cromosomas Humanos Par 14/genética , Epigenoma , Cromosomas Humanos Par 4/genética , Carbono/metabolismo , Línea Celular Tumoral , Proteínas RepresorasRESUMEN
Venetoclax is a highly potent, selective BCL2 inhibitor capable of inducing apoptosis in cells dependent on BCL2 for survival. Most myeloma is MCL1-dependent; however, a subset of myeloma enriched for translocation t(11;14) is codependent on BCL2 and thus sensitive to venetoclax. The biology underlying this heterogeneity remains poorly understood. We show that knockdown of cyclin D1 does not induce resistance to venetoclax, arguing against a direct role for cyclin D1 in venetoclax sensitivity. To identify other factors contributing to venetoclax response, we studied a panel of 31 myeloma cell lines and 25 patient samples tested for venetoclax sensitivity. In cell lines, we corroborated our previous observation that BIM binding to BCL2 correlates with venetoclax response and further showed that knockout of BIM results in decreased venetoclax sensitivity. RNA-sequencing analysis identified expression of B-cell genes as enriched in venetoclax-sensitive myeloma, although no single gene consistently delineated sensitive and resistant cells. However, a panel of cell surface makers correlated well with ex vivo prediction of venetoclax response in 21 patient samples and may serve as a biomarker independent of t(11;14). Assay for transposase-accessible chromatin sequencing of myeloma cell lines also identified an epigenetic program in venetoclax-sensitive cells that was more similar to B cells than that of venetoclax-resistant cells, as well as enrichment for basic leucine zipper domain-binding motifs such as BATF. Together, these data indicate that remnants of B-cell biology are associated with BCL2 dependency and point to novel biomarkers of venetoclax-sensitive myeloma independent of t(11;14).
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Linfocitos B/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mieloma Múltiple , Sulfonamidas/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Línea Celular Tumoral , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/metabolismo , Cromosomas Humanos Par 14/genética , Cromosomas Humanos Par 14/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Translocación Genética/efectos de los fármacosRESUMEN
TET methylcytosine dioxygenases are essential for the stability and function of regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance and express the lineage-determining transcription factor Foxp3. Here, we use whole-genome analyses to show that the transcriptional program and epigenetic features (DNA modification, chromatin accessibility) of Treg cells are attenuated in the absence of Tet2 and Tet3. Conversely, the addition of the TET activator vitamin C during TGFß-induced iTreg cell differentiation in vitro potentiates the expression of Treg signature genes and alters the epigenetic landscape to better resemble that of Treg cells generated in vivo. Vitamin C enhances IL-2 responsiveness in iTreg cells by increasing IL2Rα expression, STAT5 phosphorylation, and STAT5 binding, mimicking the IL-2/STAT5 dependence of Treg cells generated in vivo. In summary, TET proteins play essential roles in maintaining Treg molecular features and promoting their dependence on IL-2. TET activity during endogenous Treg development and potentiation of TET activity by vitamin C during iTreg differentiation are necessary to maintain the transcriptional and epigenetic features of Treg cells.
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Dioxigenasas , Linfocitos T Reguladores , Diferenciación Celular/genética , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
In both humans and mice, CTCF-binding elements form a series of interacting loops across the MHC class II (MHC-II) locus, and CTCF is required for maximal MHC-II gene expression. In humans, a CTCF-bound chromatin insulator termed XL9 and a super enhancer (SE) DR/DQ-SE situated in the intergenic region between HLA-DRB1 and HLA-DQA1 play critical roles in regulating MHC-II expression. In this study, we identify a similar SE, termed IA/IE-SE, located between H2-Eb1 and H2-Aa of the mouse that contains a CTCF site (C15) and a novel region of high histone H3K27 acetylation. A genetic knockout of C15 was created and its role on MHC-II expression tested on immune cells. We found that C15 deletion did not alter MHC-II expression in B cells, macrophages, and macrophages treated with IFN-γ because of functional redundancy of the remaining MHC-II CTCF sites. Surprisingly, embryonic fibroblasts derived from C15-deleted mice failed to induce MHC-II gene expression in response to IFN-γ, suggesting that at least in this developmental lineage, C15 was required. Examination of the three-dimensional interactions with C15 and the H2-Eb1 and H2-Aa promoters identified interactions within the novel region of high histone acetylation within the IA/IE-SE (termed N1) that contains a PU.1 binding site. CRISPR/Cas9 deletion of N1 altered chromatin interactions across the locus and resulted in reduced MHC-II expression. Together, these data demonstrate the functional redundancy of the MHC-II CTCF elements and identify a functionally conserved SE that is critical for maximal expression of MHC-II genes.
Asunto(s)
Factor de Unión a CCCTC/genética , Genes MHC Clase II/genética , Cadenas alfa de HLA-DQ/genética , Cadenas HLA-DRB1/genética , Animales , Factor de Unión a CCCTC/inmunología , Genes MHC Clase II/inmunología , Cadenas alfa de HLA-DQ/inmunología , Cadenas HLA-DRB1/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
During prolonged exposure to Ags, such as chronic viral infections, sustained TCR signaling can result in T cell exhaustion mediated in part by expression of programmed cell death-1 (PD-1) encoded by the Pdcd1 gene. In this study, dynamic changes in histone H3K4 modifications at the Pdcd1 locus during ex vivo and in vivo activation of CD8 T cells suggested a potential role for the histone H3 lysine 4 demethylase LSD1 in regulating PD-1 expression. CD8 T cells lacking LSD1 expressed higher levels of Pdcd1 mRNA following ex vivo stimulation as well as increased surface levels of PD-1 during acute, but not chronic, infection with lymphocytic choriomeningitis virus (LCMV). Blimp-1, a known repressor of PD-1, recruited LSD1 to the Pdcd1 gene during acute, but not chronic, LCMV infection. Loss of DNA methylation at Pdcd1's promoter-proximal regulatory regions is highly correlated with its expression. However, following acute LCMV infection, in which PD-1 expression levels return to near baseline, LSD1-deficient CD8 T cells failed to remethylate the Pdcd1 locus to the levels of wild-type cells. Finally, in a murine melanoma model, the frequency of PD-1-expressing tumor-infiltrating LSD1-deficient CD8 T cells was greater than in wild type. Thus, LSD1 is recruited to the Pdcd1 locus by Blimp-1, downregulates PD-1 expression by facilitating the removal of activating histone marks, and is important for remethylation of the locus. Together, these data provide insight into the complex regulatory mechanisms governing T cell immunity and regulation of a critical T cell checkpoint gene.
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Linfocitos T CD8-positivos/inmunología , Histona Demetilasas/metabolismo , Coriomeningitis Linfocítica/metabolismo , Virus de la Coriomeningitis Linfocítica/fisiología , Melanoma/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Acetilación , Enfermedad Aguda , Animales , Enfermedad Crónica , Regulación de la Expresión Génica , Histona Demetilasas/genética , Histonas/metabolismo , Activación de Linfocitos/genética , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales , Receptor de Muerte Celular Programada 1/genética , Transducción de SeñalRESUMEN
Collective invasion, the coordinated movement of cohesive packs of cells, has become recognized as a major mode of metastasis for solid tumors. These packs are phenotypically heterogeneous and include specialized cells that lead the invasive pack and others that follow behind. To better understand how these unique cell types cooperate to facilitate collective invasion, we analyzed transcriptomic sequence variation between leader and follower populations isolated from the H1299 non-small cell lung cancer cell line using an image-guided selection technique. We now identify 14 expressed mutations that are selectively enriched in leader or follower cells, suggesting a novel link between genomic and phenotypic heterogeneity within a collectively invading tumor cell population. Functional characterization of two phenotype-specific candidate mutations showed that ARP3 enhances collective invasion by promoting the leader cell phenotype and that wild-type KDM5B suppresses chain-like cooperative behavior. These results demonstrate an important role for distinct genetic variants in establishing leader and follower phenotypes and highlight the necessity of maintaining a capacity for phenotypic plasticity during collective cancer invasion.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Invasividad Neoplásica/genética , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Heterogeneidad Genética , Genómica , Humanos , Neoplasias Pulmonares/patología , Microscopía , Invasividad Neoplásica/patología , RNA-SeqRESUMEN
B cells undergo epigenetic remodeling as they differentiate into Ab-secreting cells (ASC). LSD1 is a histone demethylase known to decommission active enhancers and cooperate with the ASC master regulatory transcription factor Blimp-1. The contribution of LSD1 to ASC formation is poorly understood. In this study, we show that LSD1 is necessary for proliferation and differentiation of mouse naive B cells (nB) into plasmablasts (PB). Following LPS inoculation, LSD1-deficient hosts exhibited a 2-fold reduction of splenic PB and serum IgM. LSD1-deficient PB exhibited derepression and superinduction of genes involved in immune system processes; a subset of these being direct Blimp-1 target-repressed genes. Cell cycle genes were globally downregulated without LSD1, which corresponded to a decrease in the proliferative capacity of LSD1-deficient activated B cells. PB lacking LSD1 displayed increased histone H3 lysine 4 monomethylation and chromatin accessibility at nB active enhancers and the binding sites of transcription factors Blimp-1, PU.1, and IRF4 that mapped to LSD1-repressed genes. Together, these data show that LSD1 is required for normal in vivo PB formation, distinguish LSD1 as a transcriptional rheostat and epigenetic modifier of B cell differentiation, and identify LSD1 as a factor responsible for decommissioning nB active enhancers.
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Linfocitos B/citología , Diferenciación Celular/inmunología , Histona Demetilasas/inmunología , Células Plasmáticas/citología , Animales , Linfocitos B/inmunología , Proliferación Celular/fisiología , Ratones , Células Plasmáticas/inmunologíaRESUMEN
Epigenetic remodeling is required during B cell differentiation. However, little is known about the direct functions of epigenetic enzymes in Ab-secreting cells (ASC) in vivo. In this study, we examined ASC differentiation independent of T cell help and germinal center reactions using mice with inducible or B cell-specific deletions of Ezh2 Following stimulation with influenza virus or LPS, Ezh2-deficient ASC poorly proliferated and inappropriately maintained expression of inflammatory pathways, B cell-lineage transcription factors, and Blimp-1-repressed genes, leading to fewer and less functional ASC. In the absence of EZH2, genes that normally gained histone H3 lysine 27 trimethylation were dysregulated and exhibited increased chromatin accessibility. Furthermore, EZH2 was also required for maximal Ab secretion by ASC, in part due to reduced mitochondrial respiration, impaired glucose metabolism, and poor expression of the unfolded-protein response pathway. Together, these data demonstrate that EZH2 is essential in facilitating epigenetic changes that regulate ASC fate, function, and metabolism.
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Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Proteína Potenciadora del Homólogo Zeste 2/genética , Activación de Linfocitos/inmunología , Transcripción Genética/genética , Animales , Formación de Anticuerpos/genética , Linfocitos B/citología , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Proliferación Celular , Cromatina/fisiología , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética/genética , Centro Germinal/inmunología , Histonas/metabolismo , Lipopolisacáridos/inmunología , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Orthomyxoviridae/inmunología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genéticaRESUMEN
Expression of programmed death 1 (PD-1) on CD8 T cells promotes T cell exhaustion during chronic Ag exposure. During acute infections, PD-1 is transiently expressed and has the potential to modulate CD8 T cell memory formation. Conserved region C (CR-C), a promoter proximal cis-regulatory element that is critical to PD-1 expression in vitro, responds to NFATc1, FoxO1, and/or NF-κB signaling pathways. Here, a CR-C knockout mouse was established to determine its role on PD-1 expression and the corresponding effects on T cell function in vivo. Deletion of CR-C decreased PD-1 expression on CD4 T cells and Ag-specific CD8 T cells during acute and chronic lymphocytic choriomeningitis virus challenges, but did not affect the ability to clear an infection. Following acute lymphocytic choriomeningitis virus infection, memory CD8 T cells in the CR-C knockout mouse were formed in greater numbers, were more functional, and were more effective at responding to a melanoma tumor than wild-type memory cells. These data implicate a critical role for CR-C in governing PD-1 expression, and a subsequent role in guiding CD8 T cell differentiation. The data suggest the possibility that titrating PD-1 expression during CD8 T cell activation could have important ramifications in vaccine development and clinical care.
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Linfocitos T CD8-positivos/citología , Regulación de la Expresión Génica/inmunología , Memoria Inmunológica/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Regiones Promotoras Genéticas/genética , Animales , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Citometría de Flujo , Memoria Inmunológica/genética , Coriomeningitis Linfocítica/inmunología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Muerte Celular Programada 1/biosíntesis , Receptor de Muerte Celular Programada 1/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The oncogenic transcription factor MYC and its binding partner MAX regulate gene expression by binding to DNA at enhancer-box (E-box) elements 5Î-CACGTG-3Î. In mammalian genomes, the central E-box CpG has the potential to be methylated at the 5-position of cytosine (5mC), or to undergo further oxidation to the 5-hydroxymethyl (5hmC), 5-formyl (5fC), or 5-carboxyl (5caC) forms. We find that MAX exhibits the greatest affinity for a 5caC or unmodified C-containing E-box, and much reduced affinities for the corresponding 5mC, 5hmC or 5fC forms. Crystallization of MAX with a 5caC modified E-box oligonucleotide revealed that MAX Arg36 recognizes 5caC using a 5caC-Arg-Guanine triad, with the next nearest residue to the carboxylate group being Arg60. In an analysis of >800 primary multiple myelomas, MAX alterations occurred at a frequency of â¼3%, more than half of which were single nucleotide substitutions affecting a basic clamp-like interface important for DNA interaction. Among these, arginines 35, 36 and 60 were the most frequently altered. In vitro binding studies showed that whereas mutation of Arg36 (R36W) or Arg35 (R35H/L) completely abolished DNA binding, mutation of Arg60 (R60Q) significantly reduced DNA binding, but retained a preference for the 5caC modified E-box. Interestingly, MAX alterations define a subset of myeloma patients with lower MYC expression and a better overall prognosis. Together these data indicate that MAX can act as a direct epigenetic sensor of E-box cytosine modification states and that local CpG modification and MAX variants converge to modulate the MAX-MYC transcriptional network.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Citosina/análogos & derivados , Elementos E-Box , Mieloma Múltiple/genética , Proteínas Represoras/química , Proteínas Represoras/genética , Arginina/química , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Islas de CpG , Citosina/química , Citosina/metabolismo , ADN/química , ADN/metabolismo , Epigénesis Genética , Mutación , Unión Proteica , Conformación Proteica , Proteínas Represoras/metabolismoAsunto(s)
Mieloma Múltiple , Adrenérgicos , Células Madre Hematopoyéticas , Humanos , Mieloma Múltiple/terapiaRESUMEN
The class II transactivator (CIITA) is essential for the expression of major histocompatibility complex class II (MHC-II) genes; however, the role of CIITA in gene regulation outside of MHC-II biology is not fully understood. To comprehensively map CIITA-bound loci, ChIP-seq was performed in the human B lymphoblastoma cell line Raji. CIITA bound 480 sites, and was significantly enriched at active promoters and enhancers. The complexity of CIITA transcriptional regulation of target genes was analyzed using a combination of CIITA-null cells, including a novel cell line created using CRISPR/Cas9 tools. MHC-II genes and a few novel genes were regulated by CIITA; however, most other genes demonstrated either diminished or no changes in the absence of CIITA. Nearly all CIITA-bound sites were within regions containing accessible chromatin, and CIITA's presence at these sites was associated with increased histone H3K27 acetylation, suggesting that CIITA's role at these non-regulated loci may be to poise the region for subsequent regulation. Computational genome-wide modeling of the CIITA bound XY box motifs provided constraints for sequences associated with CIITA-mediated gene regulation versus binding. These data therefore define the CIITA regulome in B cells and establish sequence specificities that predict activity for an essential regulator of the adaptive immune response.
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Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Inmunidad Adaptativa/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Sistemas CRISPR-Cas , Línea Celular , Inmunoprecipitación de Cromatina , Secuencia Conservada , Regulación de la Expresión Génica , Genes MHC Clase II , Genoma Humano , Histonas/metabolismo , Humanos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/deficiencia , Transactivadores/genéticaRESUMEN
DNA methylation (DNAm) plays diverse roles in human biology, but this dynamic epigenetic mark remains far from fully characterized. Although earlier studies uncovered loci that undergo age-associated DNAm changes in adults, little is known about such changes during childhood. Despite profound DNAm plasticity during embryogenesis, monozygotic twins show indistinguishable childhood methylation, suggesting that DNAm is highly coordinated throughout early development. Here we examine the methylation of 27,578 CpG dinucleotides in peripheral blood DNA from a cross-sectional study of 398 boys, aged 3-17 yr, and find significant age-associated changes in DNAm at 2078 loci. These findings correspond well with pyrosequencing data and replicate in a second pediatric population (N = 78). Moreover, we report a deficit of age-related loci on the X chromosome, a preference for specific nucleotides immediately surrounding the interrogated CpG dinucleotide, and a primary association with developmental and immune ontological functions. Meta-analysis (N = 1158) with two adult populations reveals that despite a significant overlap of age-associated loci, most methylation changes do not follow a lifelong linear pattern due to a threefold to fourfold higher rate of change in children compared with adults; consequently, the vast majority of changes are more accurately modeled as a function of logarithmic age. We therefore conclude that age-related DNAm changes in peripheral blood occur more rapidly during childhood and are imperfectly accounted for by statistical corrections that are linear in age, further suggesting that future DNAm studies should be matched closely for age.
Asunto(s)
Islas de CpG/genética , Metilación de ADN , Perfilación de la Expresión Génica , Genoma Humano/genética , Adolescente , Adulto , Factores de Edad , Sitios de Unión/genética , Niño , Preescolar , Estudios Transversales , Humanos , Masculino , Metaanálisis como Asunto , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.