Gene expression changes in control- and shSamd14-infected R1 fetal liver
ABSTRACT: Samd14 was discovered as a novel GATA-2 target gene. Samd14 increased hematopoietic progenitor levels/activity, promoted signaling by a pathway instrumental for hematopoietic stem/progenitor cell regulation (Stem Cell Factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes A control shRNA or an shRNA targeting Samd14 was retrovirally introduced to fetal liver ex vivo cultures. Progenitor cells (CD71-, Ter119-) were isolated and analyzed from these cultures
Project description:Alas2 gene encodes the rate-limiting enzyme in heme biosynthesis. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis-elements strongly reduced GATA-1-induced Alas2 transcription, heme biosynthesis, and GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing Alas2 function in Alas2 cis-element-mutant (double mutant) cells by providing its catalytic product 5-aminolevulinic acid (5-ALA) rescued heme biosynthesis and the GATA-1-dependent genetic network. We discovered a GATA factor- and heme-dependent circuit that establishes the erythroid cell transcriptome. G1E-ER-GATA-1 WT and double mutant cells were examined. Untreated WT, beta-estradiol-treated WT, beta-estradiol-treated double-mutant, and beta-estradiol/5-ALA-treated double-mutant cells were subjected to RNA-seq.
Project description:Alternative splicing of the Pkm gene product generates the PKM1 and PKM2 isoforms of pyruvate kinase, and PKM2 expression is closely linked to embryogenesis, tissue regeneration, and cancer. To interrogate the functional requirement for PKM2 during development and tissue homeostasis, we generated germline PKM2 null mice (Pkm2-/-). Unexpectedly, despite being the primary isoform expressed in most wild-type adult tissues, we found that Pkm2-/- mice are viable and fertile. Thus, PKM2 is not required for embryonic or postnatal development. Loss of PKM2 leads to compensatory expression of PKM1 in the tissues that normally express PKM2. Strikingly, PKM2 loss leads to spontaneous development of hepatocellular carcinoma (HCC) with high penetrance that is accompanied by progressive changes in systemic metabolism characterized by altered systemic glucose homeostasis, inflammation, and hepatic steatosis. Therefore, in addition to its role in cancer metabolism, PKM2 plays a role in controlling systemic metabolic homeostasis and inflammation, thereby preventing HCC by a non-cell-autonomous mechanism. RNA was isolated from flash frozen ground whole liver tissue of 35 week old PKM2 KO and WT mice. Three independent mice from each condition were used as biological replicates.
Project description:Mutations within genes encoding spliceosomal proteins are the most common class of mutations in patients with myelodysplastic syndromes, yet it is currently not well understood how these mutations impact hematopoiesis or RNA splicing. Here we report that mutations affecting the splicing factor SRSF2 alter its normal RNA recognition activity, resulting in impaired hematopoietic differentiation and myelodysplasia. Commonly occurring SRSF2 mutations impaired wildtype SRSF2’s normal RNA-binding avidity and preference for specific exonic splicing enhancer RNA motifs. Integration of murine and human transcriptome data identified recurrent mis-splicing of key transcriptional regulators in the presence of mutant SRSF2, including promotion of a highly conserved “poison” exon of EZH2 that results in nonsense-mediated decay and contributes to impaired hematopoiesis. These data provide a mechanistic basis for the enrichment of specific mutations in spliceosomal proteins in myelodysplasia, and suggest that altered RNA recognition activity is a novel mechanism of leukemogenesis. mRNA profiles of murine model and K562 cells expressing SRSF2 WT, mutants and knockdown of SRSF2 in TF-1 cells generated by deep sequencing.
Project description:Substantial effort is currently devoted to identifying cancer-associated alterations using genomics. Here, we show that standard blood collection procedures rapidly change the transcriptional and post-transcriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways, up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms, and RNA surveillance inhibition. Affected genes include common mutational targets and thousands of other genes participating in processes such as chromatin modification, RNA splicing, T and B cell activation, and NF-κB signaling. The majority of published leukemic transcriptomes exhibit signals of this incubation-induced dysregulation, explaining up to 40% of differences in gene expression and alternative splicing between leukemias and reference normal transcriptomes. The effects of sample processing are particularly evident in pan-cancer analyses. We provide biomarkers that detect prolonged incubation of individual samples, and show that keeping blood on ice markedly reduces changes to the transcriptome. In addition to highlighting the potentially confounding effects of technical artifacts in cancer genomics data, our study emphasizes the need to survey the diversity of normal as well as neoplastic cells when characterizing tumors. This study is complemented by GSE61410: transcriptomic profiling of bone marrow cells from healthy individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from four healthy individuals, following an ex vivo incubation of variable length at either room temperature or on ice. RNA transcriptomes were measured using the Illumina HiSeq.
Project description:We report the biological function of Srsf2 in hematopoiesis in conditional knockout mouse models. Ablation of Srsf2 in the hematopoietic lineage caused embryonic lethality, and Srsf2-deficient fetal liver cells showed significantly enhanced apoptosis and decreased hematopoietic stem/progenitor cells. Induced ablation of Srsf2 in adult Mx1Cre/ Srsf2flox/flox mice upon polyinosinic:polycytidylic acid injection demonstrated a significant decrease in lineage-/Sca+/cKit+ cells in bone marrow. To reveal the functional impact of MDS-associated mutations in SRSF2, we profiled global splicing responses on an MDS-L cell line using RASL-seq, and found that the P95H missense mutation and P95 to R102 in-frame 8 amino-acid deletion caused significant changes in alternative splicing. The affected genes were enriched in cancer development and apoptosis. These findings suggest that intact Srsf2 is essential for the functional integrity of the hematopoietic system, and its mutations are likely key driver events to MDS. MDS-L cells (in triplicate) were transfected by srsf2 shRNA only, or pTRIPZ vectors containing both srsf2 shRNA and srsf2 mutants cDNA including P95H and P95 8 amino acid deletion as well as wild-type construct, followed by Dox induction. Total RNAs were extracted and been analyzed by RASL-seq.
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. Overall design: 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: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:This is a mathematical model describing the hematopoietic lineages with leukemia lineages, as controlled by end-product negative feedback inhibition. Variables include hematopoietic stem cells, progenitor cells, terminally differentiated HSCs, leukemia stem cells, and terminally differentiated leukemia stem cells.
Project description:We report sequential binding but unique functions of different Sox transcription factors during distinct stages of neural differentiation Loss of function experiment for Sox2/Sox3 in neural progenitor cells and gene expression profile for Sox11 expressing (PSA NCAM sorted) early formed neurons.
Project description:The transcription factor Runx1 is essential for the establishment of definitive hematopoiesis during embryonic development. In adult blood homeostasis, Runx1 plays a pivotal role in the maturation of lymphocytes and megakaryocytes. Furthermore, Runx1 is required for the regulation of hematopoietic stem and progenitor cell (HSPC) pools. However, how Runx1 orchestrates self-renewal and lineage choices in combination with other factors is not well understood. Here we describe a genome-scale RNAi screen to detect genes that cooperate with Runx1 in regulating HSPCs. We identify the polycomb group protein Pcgf1 as an epigenetic regulator involved in hematopoietic cell differentiation. We show that simultaneous depletion of Runx1 and Pcgf1 allows sustained self-renewal while blocking differentiation of HSPCs in vitro. We find an upregulation of HoxA cluster genes upon Pcgf1 knockdown that possibly accounts for the increase in self-renewal. Further, our data suggest that cells lacking both Runx1 and Pcgf1 are blocked at an early progenitor stage, indicating that a concerted action of the transcription factor Runx1 together with the epigenetic repressor Pcgf1 is necessary for terminal differentiation. Thus, our work discovers a genetic link between transcriptional and epigenetic regulation that is required for hematopoietic differentiation. Hematopoietic stem and precursor cells freshly isolated from mice were transduced with an shRNA targeting Pcgf1 or a control shRNA. Cells were selected with puromycin for 36 h before total mRNA was isolated.