investigation-genetic-dependencies-using-crispr-cas9-based

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Blood Center of Wisconsin

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is an aggressive subtype of ALL distinguished by stem-cell-associated and myeloid transcriptional programs. Inactivating alterations of Polycomb repressive complex 2 components are frequent in human ETP-ALL, but their functional role is largely undefined. We have studied the involvement of Ezh2 in a murine model of NRASQ61K-driven leukemia that recapitulates phenotypic and transcriptional features of ETP-ALL. Homozygous inactivation of Ezh2 cooperated with oncogenic NRASQ61K to accelerate leukemia onset. Inactivation of Ezh2 accentuated expression of genes highly expressed in human ETP-ALL and in normal murine early thymic progenitors. Moreover, we found that Ezh2 contributes to the silencing of stem-cell- and early-progenitor-cell-associated genes. Loss of Ezh2 also resulted in increased activation of STAT3 by tyrosine 705 phosphorylation. Our data mechanistically link Ezh2 inactivation to stem-cell-associated transcriptional programs and increased growth/survival signaling, features that convey an adverse prognosis in patients. 

Ezh2 Controls an Early Hematopoietic Program and Growth and Survival Signaling in Early T Cell Precursor Acute Lymphoblastic Leukemia.

Recent proteomic studies discovered histone lysines are modified by acylations beyond acetylation. These acylations derive from acyl-CoA metabolites, potentially linking metabolism to transcription. Bromodomains bind lysine acylation on histones and other nuclear proteins to influence transcription. However, the extent bromodomains bind non-acetyl acylations is largely unknown. Also unclear are the effects of neighboring post-translational modifications, especially within heavily modified histone tails. Using peptide arrays, binding assays, sucrose gradients, and computational methods, we quantified 10 distinct acylations for binding to the bromodomain and extraterminal domain (BET) family. Four of these acylations (hydroxyisobutyrylation, malonylation, glutarylation, and homocitrullination) had never been tested for bromodomain binding. We found N-terminal BET bromodomains bound acetylated and propionylated peptides, consistent with previous studies. Interestingly, all other acylations inhibited binding of the BET bromodomains to peptides and nucleosomes. To understand how context tunes bromodomain binding, effects of neighboring methylation, phosphorylation, and acylation within histone H4 tails were determined. Serine 1 phosphorylation inhibited binding of the BRD4 N-terminal bromodomain to polyacetylated H4 tails by >5-fold, whereas methylation had no effect. Furthermore, binding of BRDT and BRD4 N-terminal bromodomains to H4K5acetyl was enhanced 1.4-9.5-fold by any neighboring acylation of lysine 8, indicating a secondary H4K8acyl binding site that is more permissive of non-acetyl acylations than previously appreciated. In contrast, C-terminal BET bromodomains exhibited 9.9-13.5-fold weaker binding for polyacylated than for monoacylated H4 tails, indicating the C-terminal bromodomains do not cooperatively bind multiple acylations. These results suggest acyl-CoA levels tune or block recruitment of the BET bromodomains to histones, linking metabolism to bromodomain-mediated transcription.

Metabolically Derived Lysine Acylations and Neighboring Modifications Tune the Binding of the BET Bromodomains to Histone H4.

The precise molecular mechanism underlying the regulation of early B cell lymphopoiesis is unclear. The PLCγ signaling pathway is critical for antigen receptor-mediated lymphocyte activation, but its function in cytokine signaling is unknown. Here we show that PLCγ1/PLCγ2 double deficiency in mice blocks early B cell development at the pre-pro-B cell stage and renders B cell progenitors unresponsive to IL-7. PLCγ pathway inhibition blocks IL-7-induced activation of mTOR, but not Stat5. The PLCγ pathway activates mTOR through the DAG/PKC signaling branch, independent of the conventional Akt/TSC/Rheb signaling axis. Inhibition of PLCγ/PKC-induced mTOR activation impairs IL-7-mediated B cell development. PLCγ1/PLCγ2 double-deficient B cell progenitors have reduced expression of genes related to B cell lineage, IL-7 signaling, and cell cycle. Thus, IL-7 receptor controls early B lymphopoiesis through activation of mTOR via PLCγ/DAG/PKC signaling, not via Akt/Rheb signaling.

Nan Zhu

Blood Research Institute

Blood Center of Wisconsin

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Nan Zhu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Blood Research Institute

Blood Center of Wisconsin

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Blood Research Institute