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.
Project description:We have carried out high-throughput profiling of histone modifications and transcription factors in mammalian cells. First we generated stable PHF6 knock out lines by CRISPR-Cas9 technology in wild type Jurkat cells. We electroporated the RNP complex in Jurkat cells conatining guide RNAs against either non-targeting (EV) or PHF6 (KO) followed by single clone isolation and expansion. We confirmed absolute loss of total protein by WB and indel formation (mutation efficiency) at the PHF6 genomic locus by Sanger sequencing. By obtaining over six to ten billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of well studied histone modifications and transcription factors. This study provides a framework for eluciadting the role of PHF6 in regulating the chromatin landscape encompassing both activating and repressive histone modifications and chromatin remodelers in the context of leukemia.
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.
