RESUMO
Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3' splice sites (3'SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3'SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology. A synthetic minigene library with degenerate regions in 3' intronic regions (3.4 million individual minigenes) was used to compare BP usage of SF3B1K700E and SF3B1WT. Using this model, we show that SF3B1K700E spliceosomes utilize non-canonical sequence variants (at position -1 relative to BP adenosine) more frequently than wild-type spliceosomes. These predictions were confirmed using minigene splicing assays. Our results suggest a model of BP utilization by mutant SF3B1 wherein it is able to utilize non-consensus alternative BP sequences by stabilizing weaker U2-BP interactions.
Assuntos
Fatores de Processamento de RNA/metabolismo , Animais , Pareamento de Bases , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , Camundongos , Mutação , Motivos de Nucleotídeos , Fosfoproteínas/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , RNA Mensageiro/metabolismoRESUMO
Acute myeloid leukemia (AML) is driven by mutations that occur in numerous combinations. A better understanding of how mutations interact with one another to cause disease is critical to developing targeted therapies. Approximately 50% of patients that harbor a common mutation in NPM1 (NPM1cA) also have a mutation in the cohesin complex. As cohesin and Npm1 are known to regulate gene expression, we sought to determine how cohesin mutation alters the transcriptome in the context of NPM1cA. We utilized inducible Npm1cAflox/+ and core cohesin subunit Smc3flox/+ mice to examine AML development. While Npm1cA/+;Smc3Δ/+ mice developed AML with a similar latency and penetrance as Npm1cA/+ mice, RNA-seq suggests that the Npm1cA/+; Smc3Δ/+ mutational combination uniquely alters the transcriptome. We found that the Rac1/2 nucleotide exchange factor Dock1 was specifically upregulated in Npm1cA/+;Smc3Δ/+ HSPCs. Knockdown of Dock1 resulted in decreased growth and adhesion and increased apoptosis only in Npm1cA/+;Smc3Δ/+ AML. Higher Rac activity was also observed in Npm1cA/+;Smc3Δ/+ vs. Npm1cA/+ AMLs. Importantly, the Dock1/Rac pathway is targetable in Npm1cA/+;Smc3Δ/+ AMLs. Our results suggest that Dock1/Rac represents a potential target for the treatment of patients harboring NPM1cA and cohesin mutations and supports the use of combinatorial genetics to identify novel precision oncology targets.
Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Animais , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Camundongos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Medicina de Precisão , Fatores de Transcrição/genética , Proteínas rac de Ligação ao GTP , Coesinas , Proteína RAC2 de Ligação ao GTPRESUMO
Two studies investigated the effects of a live, collaborative Professional Development (PD) program versus individualized PD with a multimedia software program. For both studies, teachers were randomly assigned to either a Virtual Workshop (VW) group that used the software program or to an Actual Workshop (AW) group that participated in a face-to-face workshop that included collaborative activities. The same teaching routine, the Concept Anchoring Routine, was taught to the teachers in both studies. In Study 1, teachers' scores on a knowledge test about the routine and written plans for using the routine significantly improved from pretest to posttest in both groups. The groups' posttest scores were not significantly different. Similarly, in Study 2, both groups' posttest scores with regard to their knowledge of the routine, their written plans for use of the routine, and their implementation of the routine in their classes were significantly higher than their baseline scores. There were no differences between the teacher groups after training. The posttest knowledge scores of the whole groups of students being taught by both groups of teachers were also significantly higher than their pretest scores. Similar significant results were achieved by the students with LD. Moreover, the whole groups of students of VW teachers earned significantly higher posttest scores than the whole groups of students of AW teachers. Both teacher groups were satisfied with the training they received and with the routine. The students of both groups were satisfied with the way their teachers used the routine to help them learn.
Assuntos
Professores Escolares , Estudantes , Humanos , Aprendizagem , Instituições AcadêmicasRESUMO
Acute myeloid leukemia (AML) is a high-risk malignancy characterized by a diverse spectrum of somatic genetic alterations. The mechanisms by which these mutations contribute to leukemia development and how this informs the use of targeted therapies is critical to improving outcomes for patients. Importantly, how to target loss-of-function mutations has been a critical challenge in precision medicine. Heterozygous inactivating mutations in cohesin complex genes contribute to AML in adults by increasing the self-renewal capacity of hematopoietic stem and progenitor cells (HSPCs) by altering PRC2 targeting to induce HOXA9 expression, a key self-renewal transcription factor. Here we sought to delineate the epigenetic mechanism underpinning the enhanced self-renewal conferred by cohesin-haploinsufficiency. First, given the substantial difference in the mutational spectrum between pediatric and adult AML patients, we first sought to identify if HOXA9 was also elevated in children. Next, using primary HSPCs as a model we demonstrate that abnormal self-renewal due to cohesin loss is blocked by DOT1L inhibition. In cohesin-depleted cells, DOT1L inhibition is associated with H3K79me2 depletion and a concomitant increase in H3K27me3. Importantly, we find that there are cohesin-dependent gene expression changes that promote a leukemic profile, including HoxA overexpression, that are preferentially reversed by DOT1L inhibition. Our data further characterize how cohesin mutations contribute to AML development, identifying DOT1L as a potential therapeutic target for adult and pediatric AML patients harboring cohesin mutations.
Assuntos
Proteínas de Ciclo Celular/genética , Autorrenovação Celular , Proteínas Cromossômicas não Histona/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia Mieloide Aguda/metabolismo , Animais , Benzimidazóis/farmacologia , Proteínas de Ciclo Celular/deficiência , Células Cultivadas , Proteínas Cromossômicas não Histona/deficiência , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , CoesinasRESUMO
Tat-interactive protein 60 (Tip60) is a member of the MYST family, proteins of which are related by an atypical histone acetyltransferase (HAT) domain. Although Tip60 has been implicated in cellular activities including DNA repair, apoptosis, and transcriptional regulation, its function during embryonic development is unknown. We ablated the Tip60 gene (Htatip) from the mouse by replacing exons 1-9 with a neomycin resistance cassette. Development and reproduction of wild-type and heterozygous animals were normal. However, homozygous ablation of the Tip60 gene caused embryolethality near the blastocyst stage of development, as evidenced by inability of cells in Tip60-null blastocysts to hatch and survive in culture. Monitoring cell proliferation and death by detecting EdU-substituted DNA and TUNEL labeling revealed suppression of cell proliferation concomitant with increased cell death as Tip60-null cells attempted to hatch from blastocysts. These findings indicate that Tip60 is essential for cellular survival during the blastocyst-gastrula transition of embryogenesis.
Assuntos
Apoptose/genética , Proliferação de Células , Perda do Embrião/genética , Histona Acetiltransferases/metabolismo , Animais , Apoptose/fisiologia , Blastocisto/citologia , Blastocisto/enzimologia , Embrião de Mamíferos/enzimologia , Éxons/genética , Gástrula/enzimologia , Genótipo , Heterozigoto , Histona Acetiltransferases/genética , Homozigoto , Lisina Acetiltransferase 5 , Camundongos , Mórula/metabolismo , TransativadoresRESUMO
Acute Myeloid Leukemia (AML) is a hematologic malignancy with a poor prognosis. Recent genome-wide sequencing studies have identified frequent mutations in genes encoding members of the cohesin complex. Mutations in cohesin contribute to myeloid malignancies by conferring enhanced self-renewal of hematopoietic stem and progenitor cells but the mechanisms behind this phenotype have not been fully elucidated. Of note, cohesin mutations are highly prevalent in acute megakaryocytic leukemia associated with Down syndrome (DS-AMKL), where they occur in over half of patients. Evidence suggests that cohesin mutations alter gene expression through changes in chromatin accessibility and/or aberrant targeting of epigenetic complexes. In this review we discuss the pathogenic mechanisms by which cohesin mutations contribute to myeloid malignancies.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Leucemia Mieloide Aguda/genética , Humanos , Mutação , CoesinasRESUMO
Tat-interactive protein 60 (Tip60), encoded by the Kat5 gene, is a member of the MYST family of acetyltransferases. Cancer biology studies have shown that Tip60 induces the DNA damage response, apoptosis, and cell-cycle inhibition. Although Tip60 is expressed in the myocardium, its role in cardiomyocytes (CMs) is unclear. Earlier studies here showed that application of cardiac stress to globally targeted Kat5+/-haploinsufficient mice resulted in inhibition of apoptosis and activation of the CM cell-cycle, despite only modest reduction of Tip60 protein levels. It was therefore of interest to ascertain the effects of specifically and substantially depleting Tip60 from CMs using Kat5LoxP/-;Myh6-Cre mice in the absence of stress. We report initial findings using this model, in which the effects of specifically depleting Tip60 protein from ventricular CMs, beginning at early neonatal stages, were assessed in 2-12 week-old mice. Although 5'-bromodeoxyuridine immunostaining indicated that CM proliferation was not altered at any of these stages, CM density was increased in 2 week-old ventricles, which persisted in 4 week-old hearts when TUNEL staining revealed inhibition of apoptosis. By week 4, levels of connexin-43 were depleted, and its patterning was dysmorphic, concomitant with an increase in cardiac hypertrophy marker expression and interstitial fibrosis. This was followed by systolic dysfunction at 8 weeks, after which extensive apoptosis and CM fallout occurred, followed by lethality as mice approached 12 weeks of age. In summary, chronic depletion of Tip60 from the ventricular myocardium beginning at early stages of neonatal heart development causes CM death after 8 weeks; hence, Tip60 protein has a crucial function in the heart.
Assuntos
Coração/fisiopatologia , Histona Acetiltransferases/fisiologia , Miócitos Cardíacos/metabolismo , Transativadores/fisiologia , Alelos , Animais , Lisina Acetiltransferase 5 , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Tat-interactive protein 60 (Tip60) is a member of the MYST family of histone acetyltransferases. Studies using cultured cells have shown that Tip60 has various functions including DNA repair, apoptosis and cell-cycle regulation. We globally ablated the Tip60 gene (Htatip), observing that Tip60-null embryos die at the blastocyst stage (Hu et al. Dev.Dyn.238:2912;2009). Although adult heterozygous (Tip60(+/-)) mice reproduce normally without a haploinsufficient phenotype, stress caused by Myc over-expression induced B-cell lymphoma in Tip60(+/-) adults, suggesting that Tip60 is a tumor suppressor (Gorrini et al. Nature 448:1063;2007). These findings prompted assessment of whether Tip60, alternative splicing of which generates two predominant isoforms termed Tip60α and Tip60ß, functions to suppress the cell-cycle in adult cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting revealed that Tip60α is the predominant Tip60 isoprotein in the embryonic heart, transitioning at neonatal stages to Tip60ß, which is the only isoprotein in the adult heart wherein it is highly enriched. Over-expression of Tip60ß, but not Tip60α, inhibited cell proliferation in NIH3T3 cells; and, Tip60-haploinsufficient cultured neonatal cardiomyocytes exhibited increased cell-cycle activity. To address whether Tip60ß suppresses the cardiomyocyte cell-cycle in the adult heart, hypertrophic stress was induced in Tip60(+/+) and Tip(+/-) littermates via two methods, Myc over-expression and aortic banding. Based on immunostaining cell-cycle markers and western blotting cyclin D, stress increased cardiomyocyte cell-cycle mobilization in Tip60(+/-) hearts, in comparison with Tip60(+/+) littermates. Aortic-banded Tip60(+/-) hearts also exhibited significantly decreased apoptosis. CONCLUSIONS/SIGNIFICANCE: These findings provide evidence that Tip60 may function in a tumor suppressor pathway(s) to maintain adult cardiomyocytes in replicative senescence.