RESUMO
Recombinant adeno-associated virus (rAAV) vectors have the unique property of being able to perform genomic targeted integration (TI) without inducing a double-strand break (DSB). In order to improve our understanding of the mechanism behind TI mediated by AAV and improve its efficiency, we performed an unbiased genetic screen in human cells using a promoterless AAV-homologous recombination (AAV-HR) vector system. We identified that the inhibition of the Fanconi anemia complementation group M (FANCM) protein enhanced AAV-HR-mediated TI efficiencies in different cultured human cells by â¼6- to 9-fold. The combined knockdown of the FANCM and two proteins also associated with the FANCM complex, RecQ-mediated genome instability 1 (RMI1) and Bloom DNA helicase (BLM) from the BLM-topoisomerase IIIα (TOP3A)-RMI (BTR) dissolvase complex (RMI1, having also been identified in our screen), led to the enhancement of AAV-HR-mediated TI up to â¼17 times. AAV-HR-mediated TI in the presence of a nuclease (CRISPR-Cas9) was also increased by â¼1.5- to 2-fold in FANCM and RMI1 knockout cells, respectively. Furthermore, knockdown of FANCM in human CD34+ hematopoietic stem and progenitor cells (HSPCs) increased AAV-HR-mediated TI by â¼3.5-fold. This study expands our knowledge on the mechanisms related to AAV-mediated TI, and it highlights new pathways that might be manipulated for future improvements in AAV-HR-mediated TI.
Assuntos
Sistemas CRISPR-Cas , DNA Helicases/antagonistas & inibidores , Proteínas de Ligação a DNA/antagonistas & inibidores , Dependovirus/genética , Edição de Genes , Células-Tronco Hematopoéticas/metabolismo , RecQ Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Vetores Genéticos , Células HeLa , Células-Tronco Hematopoéticas/citologia , Recombinação Homóloga , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is implicated in myeloid malignancies with deletion of chromosome 5q. Employing a combination of proteomic and genetic approaches, we find that TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. Expression of TIFAB in hematopoietic stem/progenitor cells (HSPCs) permits USP15 signaling to substrates, including MDM2 and KEAP1, and mitigates p53 expression. Consequently, TIFAB-deficient HSPCs exhibit compromised USP15 signaling and are sensitized to hematopoietic stress by derepression of p53. In MLL-AF9 leukemia, deletion of TIFAB increases p53 signaling and correspondingly decreases leukemic cell function and development of leukemia. Restoring USP15 expression partially rescues the function of TIFAB-deficient MLL-AF9 cells. Conversely, elevated TIFAB represses p53, increases leukemic progenitor function, and correlates with MLL gene expression programs in leukemia patients. Our studies uncover a function of TIFAB as an effector of USP15 activity and rheostat of p53 signaling in stressed and malignant HSPCs.
Assuntos
Hematopoese , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Domínio Catalítico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Ligação Proteica , Estresse Fisiológico , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/química , UbiquitinaçãoRESUMO
TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML.
