Project description:ZNF384-rearranged fusion oncoproteins (FO) define a subset of lineage ambiguous leukemias, but the mechanistic role of ZNF384 FO in leukemogenesis and lineage ambiguity is poorly understood. Here, using viral expression in mouse and human hematopoietic stem and progenitor cells (HSPCs) and a Ep300-Zfp384 mouse model we show that ZNF384 FO promote hematopoietic expansion, myeloid lineage skewing, and self-renewal. In mouse HSPCs, concomitant lesions such as NRASG12D, were required for fully penetrant leukemia, whereas expression of ZNF384 FO drove development of B/myeloid leukemia in human HSPCs, with sensitivity of human ZNF384r leukemia to FLT3 inhibition in vivo. Mechanistically, ZNF384 FO occupy a subset of predominantly intragenic/enhancer regions with increased histone 3 lysine acetylation suggesting enhancer function. These data define a paradigm for FO-driven lineage ambiguous leukemia, in which expression in HSPCs results in deregulation of lineage-specific genes and hematopoietic skewing, progressing to full leukemic transformation in the presence of proliferative stress.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.
Project description:Mx1-Cre/KrasG12D mice were injected with pIpC to induce a myeloproliferative disease resembling human chronic myelomonocytic leukemia (CMML-like MPD). When the disease was fully developed, CD11b-/Ly6G-/c-Kit+ hematopoietic stem and progenitor cells (HSPCs) were isolated and subjected to miR-microarray expression profiling. pIpC-injected CD11b-/Ly6G-/c-Kit+ HSPCs of age matched wildytpe control mice were used as controls.
Project description:Increased expression of Kruppel like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7-/- cells was comparable to control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, while resulting in multi-lineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21Cip1/Waf1) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor (HSPC) function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival. KLF7 overexpression in HSPCs expression array: Lin- c-Kit+ Sca-1+ cells transduced with a KLF7 expressing or control (empty vector) lentivirus. Expression profiles of KLF7 overexpressing vs controls HSPCs. Cells were harvested 72 hrs post-transduction to compare expression profiles of control vs KLF7 overexpressing HSPCs
Project description:To better understand the early events regulating lineage-specific hematopoietic differentiation, we analyzed the transcriptional profiles of CD34+ human hematopoietic stem and progenitor cells (HSPCs) subjected to differentiation stimulus. CD34+ cells were cultured for 12 and 40 hours in liquid cultures with supplemented media favoring myeloid or erythroid commitment. Serial analysis of gene expression (SAGE) was employed to generate four independent libraries. CD34+ Hematopoietic Stem Progenitor Cells with no differentiation stimulus were used as a control library.
Project description:IRF2, IRF6, and MYB are candidate regulators of human erythropoiesis. We here examine primary CD34+ hematopoietic stem/progenitor cells (HSPCs)-derived erythroid progenitors with control, IRF2, IRF6, or MYB shRNA lentiviral transduction prior to differentiation. Gene expression microarray profiling datasets for MYB shRNA and control shRNA were obtained from Gene Expression Omnibus (GEO) under accession number GSE25678. The data were analyzed together with the datasets obtained in this study. Primary maturing adult erythroblasts were generated ex vivo from CD34+ hematopoietic stem/progenitor cells (HSPCs) using a serum-free two-phase liquid culture system. CD34+ HSPCs were transduced with lentiviruses containing shRNAs against IRF2 or IRF6 gene, selected and differentiated to proerythroblasts (ProEs). Cells were harvested at day 5 of differentiated and total RNA were extracted. This was used to hybridize to Affymetrix expression arrays using the HG-U133 Plus 2.0 platform.
Project description:Obesity is known to affect bone marrow hematopoietic stem and progenitor cells (HSPCs). We used single cell RNA sequencing (scRNA-seq) to analyze the differences in HSPCs isolated from lean and obese mice.