ChIP-seq of CD117-positive primary hematopoietic precursor cell from Meer mice that carry a leukemogenic knock-in of an inducible ENL-ER portion into the mouse Mll locus
ABSTRACT: This ChIP-seq experiment was done to investigate the global distribution of H2A ubiquitination (a hallmark of polycomb mediated repression), H2B ubiquitination (introduced by PAF-complex associated ubiquitin ligases and connected to active transcription), and PAF1 occupation in Meer cells. Meer cells are primary cells transformed by a knock-in of an inducible Mll-ENL oncogene. ENL and ENLins ChIP-seqs were done in CD117-positive primary hematopoietic cells without any pretransformation by other oncogenes.
Project description:MLL fusions are leukemogenic transcription factors that enhance transcriptional elongation through modification of chromatin and RNAPolII. Global transcription rates and chromatin changes accompanying the transformation process were monitored by nascent-RNA-seq and ChIP-seq identifying 165 targets separated into two distinct clades. This accession contains ChIP Seq experiments for MLLENL and a negative control. The RNA-seq data set could also be found in ArrayExpress under accession number E-MTAB-3591 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3591/ ).
Project description:MLL fusions are leukemogenic transcription factors that enhance transcriptional elongation through modification of chromatin and RNAPolII. Global transcription rates and chromatin changes accompanying the transformation process were monitored by nascent-RNA-seq and ChIP-seq identifying 165 targets separated into two distinct clades. ChIP-seq data complementing this RNA-seq data set can be found in ArrayExpress under accession number E-MTAB-3593 (http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3593)
Project description:MLL encodes a histone methyltransferase that is critical in maintaining gene expression during embryonic development and hematopoiesis. 11q23 translocations encode chimeric MLL fusions that act as potent drivers of acute leukemia. However, it remains unclear what portion of the leukemic genome is under the direct control of the MLL fusion protein. By comparing patient-derived leukemic cell lines, we find that MLL fusion-bound genes are a small subset of that recognized by wild-type MLL. In an inducible MLL-ENL cellular model, binding of the MLL fusion protein and changes in H3K79 methylation are limited to a specific portion of the genome, whereas wild-type MLL distributes to a much larger set of gene loci. Surprisingly, among 223 MLL fusion-bound genes, only 12 demonstrate a significant increase in mRNA expression upon induction of the fusion protein. In addition to Hoxa9 and Meis1, this includes Eya1 and Six1 which comprise a heterodimeric transcription factor important in several developmental pathways. We show that Eya1 has the capacity to immortalize hematopoietic progenitor cells in vitro and collaborates with Six1 in hematopoietic transformation assays. Altogether, our data suggest that MLL fusions contribute to the development of acute leukemia through direct activation of a small set of target genes. We explored an inducible MLL-ENL cellular model, which was obtained from Dr. Robert Slany (University Erlangen, Germany). We wished to examine the differential expressed genes that are bound by MLL wild type (No 4-OHT) and fusion (4-OHT) proteins, combinding the ChIP-chip data to explore the potential MLL fusion-regulated genes.
Project description:Translocations involving the MLL genes are frequently found in Acute Myeloid Leukemia (AML) and are associated with poor prognosis. The MLL fusion proteins act as aberrant transcription factor activating a transcriptional program that transforms the cells, potentially through collaboration with other transcription factors. To investigate this we searched gene expression profiles from patients with MLL-rearranged AML compared with normal hematopoietic progenitor cells for transcriptional regulators and found targets of C/EBPα to be up-regulated in the AML samples, suggesting that C/EBPα might collaborate with MLL fusion proteins in the initial transformation process. We could show that transformation by MLL fusion proteins is dependent on C/EBPα activity both in early progenitors as well as in GMPs. In contrast, C/EBPα was found to be indispensable in an already established leukemia. These results suggest that C/EBPα play an important role in the early transforming event of leukemogenesis. We used microarray to study the early transcriptional changes induced by MLL-ENL expression and we identified a combined C/EBPα / MLL-ENL transcriptional signature. 3 Cebpaflox/flox;Mx1Cre and 3 Cebpaflox/flox;Mx1Cre- mice were sacrificed 14 days after pIpC injection and bone marrow cells were harvested, enriched for cKit-expression and transduced with a pMIG retroviral vector expressing the MLL-ENL fusion protein and GFP. 72 h post first transduction, GFP-positive or negative PreGM cells were sorted.
Project description:It is known that NK cells are a heterogeneous population of functionally distinct NK cell subsets. Here we report on different genomic, phenotypic and functional properties of four murine NK cell subsets distinguished by CD117 (c-kit), CD27 and CD11b expression. Gene expression was measured in NK cell subsets freshly sorted from murine C57Bl/6 splenocytes. Two to three different batches were analysed.
Project description:Paternal imprinting initiates in primordial germ cells (PGCs), and is considered largely completed at birth. The resulting postnatal spermatogonial stem cells (SSCs) thenself-renew and proliferate to populate the testicular niche, with sexual maturation enabling productive gametogenesis. mRNA profiles of neonatal wild type (WT) mice testis were generated by deep sequencing using Illumina HiSeq 2000 Examination of 2 different histone modifications in mouse spermatogonia Please note that ChIPSeq_Kitplus samples are samples isolated with MACS CD117 microbeads from Miltenyi and ChIPSeq_Kitminus are samples that were not positively selected for Kit.
Project description:Acute myelogenous leukemia (AML) is driven by leukemic stem cells (LSC) generated by mutations that confer (or maintain) self-renewal potential coupled to an aberrant differentiation program. Using retroviral mutagenesis, we identified genes that generate LSC in collaboration with genetic disruption of the gene encoding interferon response factor 8 (Irf8), which induces a myeloproliferation in vivo. Amongst the targeted genes, we identified Mef2c, encoding a MADS transcription factor, and confirmed that over-expression induced a myelomonocytic leukemia in cooperation with Irf8 deficiency. Strikingly, several of the genes identified in our screen have been reported to be upregulated in the mixed-lineage leukemia (MLL) subtype. High MEF2C expression levels were confirmed in AML patient samples with MLL gene disruptions, prompting an investigation of the causal interplay. Using a conditional mouse strain, we demonstrated that Mef2c deficiency does not impair the establishment nor maintenance of LSC generated in vitro by MLL/ENL fusion proteins – however, its loss led to compromised homing and invasiveness of the tumor cells. Mef2c-dependent targets included several genes encoding matrix metalloproteinases and chemokine ligands and receptors, providing a mechanistic link to increased homing and motility. Thus an early event in LSC generation may be responsible for the aggressive nature of this leukemia subtype. Experiment Overall Design: For gene expression profiling of M/E cells, RNA was isolated from M/E-Mef2c del/- cells infected with MYs-iPAC (empty vector) or MYs-pMef2cASR after puromycin selection at three different time points, pooled, and hybridized against the Agilent Whole Mouse Genome Microarray 4x44K using the one-color service of Miltenyi. Transcript levels were verified by real-time RT-PCR using the SYBRGreen Reaction Mix (Roche Mannheim) in a Roche Light-Cycler. cDNA levels were normalized against Hprt transcript levels. Primers and amplification conditions are available upon request.
Project description:Activation of non-canonical TGF-β1 signaling indicates an autoimmune mechanism for bone marrow fibrosis in primary myelofibrosis Two condition experiment. Biological replicates: 3 control human healty subjects, 6 PMF patients
Project description:The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, raising the possibility that MYB may be a therapeutic target. However realization of this potential requires (i) a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis; and (ii) an approach for developing an effective therapeutic. We previously showed that the interaction of Myb with the coactivator CBP/p300 is essential for its transforming activity. Here we use hematopoietic cells from the Booreana mouse strain, which carries a mutation in Myb that prevents interaction with CBP/p300, to examine the requirement for this interaction in myeloid transformation and leukemogenesis. Using this strain and a strain (plt6) carrying a “complementary” mutation in p300, we show that the Myb-p300 interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type (WT) mice, Booreana cells fail to induce leukemia upon transplantation into irradiated recipients following transduction with an AML1-ETO9a retrovirus. These data highlight disruption of the Myb-p300 interaction as a potential therapeutic strategy for AML and suggest that such a strategy would have a useable therapeutic index since Booreana mice, unlike Myb null mice, are viable. Finally we have begun to explore the molecular basis of the these observations by gene expression profiling; this highlighted several genes previously implicated in myeloid leukemogenesis as being differentially expressed between WT and Booreana cells transduced with AML1-ETO9a. Total RNA was obtained from FACS sorted GFP+;c-Kit+ primary bone marrow cells from WT and Booreana mouse strains which had been cultured for 48 hours post-transduction with Control or AML1-ETO9a retroviruses. RNA was extracted from each of 4 samples per group and used to probe Illumina mouse Beadchips array.
Project description:T-cell prolymphocytic leukemina (T-PLL) is an agressive lymphoma derived from mature T-cells, which is in most cases characterized by the presence of an inv(14)(q11q32) and a characteristic pattern of secondary chromosomal abberations. We used microarrays to compare the transcriptomes of eight immunomagnetically purified CD3+ normal donor derived peripheral blood cells with five highly purified inv(14)-positive T-PLL blood samples. Experiment Overall Design: Purified T-PLL cells and normal T-cells were analyzed on microarrays to identify differentially expressed genes. High-resolution copy number determination using SNP-chip technology and FISH in twelve inv(14)-positive T-PLL showed that differentially expressed genes clustered significantly in regions affectd by recurrent chromosomal imbalances.