Project description:The plant homeodomain 6 gene (PHF6) is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL); however, its specific functional role in leukemia development remains to be established. Here, we show that loss of PHF6 is an early mutational event in leukemia transformation. Mechanistically, genetic inactivation of Phf6 in the hematopoietic system enhances hematopoietic stem cell (HSC) long-term self-renewal and hematopoietic recovery after chemotherapy by rendering Phf6 knockout HSCs more quiescent and less prone to stress-induced activation. Consistent with a leukemia-initiating tumor suppressor role, inactivation of Phf6 in hematopoietic progenitors lowers the threshold for the development of NOTCH1-induced T-ALL. Moreover, loss of Phf6 in leukemia lymphoblasts activates a leukemia stem cell transcriptional program and drives enhanced T-ALL leukemia-initiating cell activity. These results implicate Phf6 in the control of HSC homeostasis and long-term self-renewal and support a role for PHF6 loss as a driver of leukemia-initiating cell activity in T-ALL.

2021-06-23 | GSE178739 | GEO

Crystal structure of Homo sapiens kynureninase.

Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.

| S-EPMC2531291 | biostudies-literature

Homo sapiens whole blood.

Project description:RNA extracted at 240min. Samples are rRNA depleted. Expression measurement of Homo sapiens genes.

2019-07-31 | GSE114179 | GEO

Phf6-null hematopoietic stem cells have enhanced self-renewal capacity and oncogenic potentials

Project description:Plant homeodomain finger gene 6 (PHF6) encodes a 365-amino-acid protein containing two plant homology domain fingers. Germline mutations of human PHF6 cause Börjeson–Forssman–Lehmann syndrome, a congenital neurodevelopmental disorder. Loss-of-function mutations of PHF6 are detected in patients with acute leukemia, mainly of T cell lineage and in a small proportion of myeloid lineage. The functions of PHF6 in physiological hematopoiesis and leukemogenesis remain incompletely defined. To address this question, we generated a conditional Phf6 knockout mouse model and investigated the impact of Phf6 loss on the hematopoietic system. We found that Phf6 knockout mice at 8-week age had reduced numbers of CD4+ and CD8+ T cells in the peripheral blood compared to the wild-type littermates. There were decreased granulocyte-monocytic progenitors but increased Lin-c-Kit+Sca-1+ (LSK) cells in the marrow of young Phf6 knockout mice. Functional studies including competitive repopulation unit and serial transplantation assays revealed an enhanced reconstitution and self-renewal capacity in Phf6 knockout hematopoietic stem cells (HSCs). Aged Phf6 knockout mice had myelodysplasia-like presentations including decreased platelet counts, megakaryocyte dysplasia, and enlarged spleen related to extramedullary hematopoiesis. Moreover, we found that Phf6 loss lowered the threshold of NOTCH1-induced leukemic transformation at least partially through increased leukemia-initiating cells. Transcriptome analysis on the restrictive rare HSC subpopulations revealed upregulated cell cycling and oncogenic functions, with alteration of expression of key genes in those pathways. In summary, our studies demonstrate the in vivo crucial roles of Phf6 in physiological and malignant hematopoiesis.

2019-08-21 | GSE129465 | GEO

HoxB4 in hematopoietic stem cells

Project description:Ectopic expression of homeodomain transcription factor HoxB4 in mouse ES cells enhances in vitro development of hematopoietic stem cells (HSCs). Mouse ES cells were differentiated for 6 days in EBs, then c-Kit+CD41+ hematopoietic precursor cells were isolated, plated onto OP9 cells, and transduced with the GFP control or 3M-CM-^WFLAG-tagged HoxB4 retrovirus. At day 12 of the co-culture with OP9 cells, c-Kit+ progenitor cells overexpressing HoxB4 were collected by magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip and microarray analysis. The purity of the recovered c-Kit+ cells was more than 95% as judged by flow cytometry.

2011-05-23 | E-GEOD-26653 | biostudies-arrayexpress