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In chronic lymphocytic leukemia (CLL), a diverse set of genetic mutations is embedded in a deregulated epigenetic landscape that drives cancerogenesis. To elucidate the role of aberrant chromatin features, we mapped DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, binding of EBF1 and CTCF, as well as the transcriptome of B cells from CLL patients and healthy donors. A globally increased histone deacetylase activity was detected and half of the genome comprised transcriptionally downregulated partially DNA methylated domains demarcated by CTCF. CLL samples displayed a H3K4me3 redistribution and nucleosome gain at promoters as well as changes of enhancer activity and enhancer linkage to target genes. A DNA binding motif analysis identified transcription factors that gained or lost binding in CLL at sites with aberrant chromatin features. These findings were integrated into a gene regulatory enhancer containing network enriched for B‐cell receptor signaling pathway components. Our study predicts novel molecular links to targets of CLL therapies and provides a valuable resource for further studies on the epigenetic contribution to the disease. Synopsis Transcriptome profiling and genome‐scale mapping of DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, EBF1 and CTCF binding are performed in B cells from CLL patients and healthy donors. Altered chromatin features were detected at 80% of the differentially regulated genes in CLL and histone deacetylase activity was globally increased. Half of the CLL genome comprised partially DNA methylated domains that were transcriptionally downregulated, demarcated by CTCF and enriched for H3K9me3 and H3K27me3. H3K4me3 was redistributed at CLL promoters, including loss of bivalent H3K4me3/H3K27me3 states, and substantial changes of enhancer activity were detected. A gene regulatory network including enhancers was constructed around the transcription factors targeting 15 central binding motifs that were associated with aberrant CLL chromatin features. Genes involved in BCR signaling were enriched in the network. Graphical Abstract Transcriptome profiling and genome‐scale mapping of DNA methylation, seven histone modifications, nucleosome positions, chromatin accessibility, EBF1 and CTCF binding are performed in B cells from CLL patients and healthy donors.

The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence 'Apoptosis induced Proliferation' (AiP) and 'Apoptosis induced Apoptosis' (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

Putzig (Pzg) was originally identified as being an integral component of the TRF2/DREF complex in Drosophila melanogaster, thereby regulating the transcriptional activation of replication-related genes. In a DREF-independent manner, Pzg was shown to mediate Notch target gene activation. This function of Pzg entails an association with the nucleosome remodeling factor complex NURF, which directly binds the ecdysone receptor EcR and coregulates targets of the EcR via the NURF-specific subunit Nurf-301. In contrast, Nurf-301 acts as a negative regulator of JAK/STAT signaling. Here, we provide evidence to show that Pzg is fundamental for these functions of NURF, apart from the regulation of Notch signaling activity. A jump-out mutagenesis provided us with a pzg null mutant displaying early larval lethality, defects in growth, and molting accompanied by aberrant feeding behavior. We show that Pzg is associated with EcR in vivo and required for the transcriptional induction of EcR target genes, whereas reduced ecdysteroid levels imply a NURF-independent function of Pzg. Moreover, pzg interferes with JAK/STAT-signaling activity by acting as a corepressor of Ken. Lamellocyte differentiation was consistently affected in a JAK/STAT mutant background and the expression level of defense response genes was elevated in pzg mutants, leading to the formation of melanotic tumors. Our results suggest that Pzg acts as an important partner of NURF in the regulation of EcR and JAK/STAT signaling.

Overexpression of Hairless (H) causes a remarkable degree of tissue loss and apoptosis during imaginal development. H functions as antagonist in the Notch-signaling pathway in Drosophila, and the link to growth and apoptosis is poorly understood. To further our insight into H-mediated apoptosis, we performed two large-scale screens for modifiers of a small rough eye phenotype caused by H overexpression. Both loss- and gain-of-function screens revealed known and new genetic interactors representing diverse cellular functions. Many of them did not cause eye phenotypes on their own, emphasizing a specific genetic interaction with H. As expected, we also identified components of different signaling pathways supposed to be involved in the regulation of cell growth and cell death. Accordingly, some of them also acted as modifiers of proapoptotic genes, suggesting a more general involvement in the regulation of apoptosis. Overall, these screens highlight the importance of H and the Notch pathway in mediating cell death in response to developmental and environmental cues and emphasize their role in maintaining developmental cellular homeostasis.

Chronic lymphocytic leukemia (CLL) is associated with substantial alterations in T-cell composition and function. However, the role of T-cells in CLL remains largely controversial. Here, we utilized the Eµ-TCL1 mouse model of CLL as well as blood and lymph node samples of CLL patients to investigate the existence of anti-tumoral immune responses in CLL, and to characterize involved immune cell populations. Thereby, we identified an oligoclonal CD8+ effector T-cell population that expands along with CLL progression and controls disease development. We further show that a higher percentage of CD8+ effector T-cells produces IFNγ, and demonstrate that neutralization of IFNγ results in faster CLL progression in mice. Phenotypical and functional analyses of expanded CD8+ effector T-cells show significant differences in disease-affected tissues in mice, with cells in secondary lymphoid organs harboring hallmarks of activation-induced T-cell exhaustion. Notably, we further describe a respective population of exhausted CD8+ T-cells that specifically accumulate in lymph nodes, but not in peripheral blood of CLL patients. Collectively, these data emphasize the non-redundant role of CD8+ T-cells in suppressing CLL progression and highlight their dysfunction that can be exploited as target of immunotherapy in this malignancy.

Chronic lymphocytic leukemia (CLL) cells depend on microenvironmental non-malignant cells for survival. We compared the transcriptomes of primary CLL cells cocultured or not with protective bone marrow stromal cells (BMSCs) and found that oxidative phosphorylation, mitochondrial function, and hypoxic signaling undergo most significant dysregulation in non-protected CLL cells, with the changes peaking at 6–8 h, directly before induction of apoptosis. A subset of CLL patients displayed a gene expression signature resembling that of cocultured CLL cells and had significantly worse progression-free and overall survival. To identify drugs blocking BMSC-mediated support, we compared the relevant transcriptomic changes to the Connectivity Map database. Correlation was found with the transcriptomic signatures of the cardiac glycoside ouabain and of the ipecac alkaloids emetine and cephaeline. These compounds were highly active against protected primary CLL cells (relative IC50's 287, 190, and 35 nM, respectively) and acted by repressing HIF-1α and disturbing intracellular redox homeostasis. We tested emetine in a murine model of CLL and observed decreased CLL cells in peripheral blood, spleen, and bone marrow, recovery of hematological parameters and doubling of median survival (31.5 vs. 15 days, P = 0.0001). Pathways regulating redox homeostasis are thus therapeutically targetable mediators of microenvironmental support in CLL cells.

The Drosophila gene putzig (pzg) encodes a nuclear protein that is an integral component of the Trf2/Dref complex involved in the transcription of proliferation-related genes. Moreover, Pzg is found in a complex together with the nucleosome remodeling factor NURF, where it promotes Notch target gene activation. Here we show that downregulation of pzg activity in the developing wing imaginal discs induces an apoptotic response, accompanied by the induction of the pro-apoptotic gene reaper, repression of Drosophila inhibitor of apoptosis protein accumulation and the activation of the caspases Drice, Caspase3 and Dcp1. As a further consequence 'Apoptosis induced Proliferation' (AiP) and 'Apoptosis induced Apoptosis' (AiA) are triggered. As expected, the activity of the stress kinase Jun N-terminal kinase (JNK), proposed to mediate both processes, is ectopically induced in response to pzg loss. In addition, the expression of the mitogen wingless (wg) but not of decapentaplegic (dpp) is observed. We present evidence that downregulation of Notch activates Dcp1 caspase and JNK signaling, however, neither induces ectopic wg nor dpp expression. In contrast, the consequences of Dref-RNAi were largely indistinguishable from pzg-RNAi with regard to apoptosis induction. Moreover, overexpression of Dref ameliorated the downregulation of pzg compatible with the notion that the two are required together to maintain cell and tissue homeostasis in Drosophila.