Project description:SEM cells were established from the peripheral blood of a 5-year-old girl in relapse with acute lymphoblastic leukaemia (ALL). SEM cells exhibit the t(4;11) chromosomal rearrangement, which leads to production of the MLL-AF4 fusion protein. Hematopoietic transcription factors including HOXA9 and MEIS1 are highly expressed in ALL. ChIP-seq was performed against HoxA9 and MEIS1 in SEM cells. DNA was enriched by chromatin immunoprecipitation (ChIP) and analyzed by Solexa sequencing.
Project description:SEM cells were established from the peripheral blood of a 5-year-old girl in relapse with acute lymphoblastic leukaemia (ALL). SEM cells exhibit the t(4;11) chromosomal rearrangement, which leads to production of the MLL-AF4 fusion protein. Hematopoietic transcription factors including HOXA9 and MEIS1 are highly expressed in ALL.
Project description:The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feed-forward loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, we show that Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.
Project description:DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we report that DNMT3A mutational âhotspotâ at Arg882 (DNMT3A-R882H) cooperates with constitutively activated RAS in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. DNMT3A-R882H potentiates aberrant transactivation of âstemnessâ gene expression programs, notably transcription factors Meis1, Hox-A, Mn1 and Mycn. Mechanistically, R882-mutated DNMT3A directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3A-R882H induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Inactivation of Dot1l represses DNMT3AR882H-mediated stem cell gene dysregulation and acute leukemogenicity. In this dataset, we provided R882H-mutated DNMT3A, H3K4me1, H3K4me3 and H3K27me3 ChIP-seq profiling data of RH-RAS LSCs, and H3K4me1 ChIP-seq data in HOXA9-MEIS1 LSCs. Genome-wide binding of R882H-mutated DNMT3A (Myc tagged; ChIP-seq with 9e10 anti-Myc antibodies) and histone modification profiles for H3K4me1, H3K4me3 and H3K27me3 were generated by ChIP-seq using specific antibodies in RH-RAS LSCs. Genome-wide H3K4me1 histone modification profiles were generated by ChIP-seq using H3K4me1 specific antibody in HOXA9-MEIS1 LSCs.
Project description:The clustered homeobox proteins play crucial roles in development, hematopoiesis and leukemia yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 co-bind at hundreds of highly evolutionarily-conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation and transcriptional activation of a network of proto-oncogenes including Erg, Flt3, Lmo2, Myb and Sox4. Collectively this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia. To identify the genome-wide binding sites for Hoxa9 and the Hox cofactor Meis1
Project description:Meis1 is found cooperatively activated with Hoxa7/a9 in AML, and it indeed promotes leukemogenic activities of Hoxa9. It is important to identify downstream target genes of Meis1 to understand its cooperative activity with Hoxa9 in leukemogenesis. We used microarrays to detail the global programme of gene expression upon Meis1 knockout. Murine primary bone marrow cells of the Rosa26-Cre-ERT2 knock-in mouse were transformed by retroviral transduction of Hoxa9 and floxed Meis1. The immortalized bone marrow cells were treated with 2 μM of 4-hydroxytamoxifen to delete Meis1 cDNA. Gene expression profiles were compared between the original Hoxa9/Meis1-expressing cells and Meis1 deleted (Hoxa9 only) cells.
Project description:HOXA9/MEIS1 plays a synergistic causative role and overexpresses frequently in acute myeloid leukemia (AML). Hoxa9/Meis1 transgenic murine results in rapid leukemic transformation of primary bone marrow cells. However, murine model is not suitable to perform a high-throughput phenotypic screen in vivo and identify compounds for AML therapy. A transgenic zebrafish overexpresses hoxa9/meis1 need to generate. We have engineered an inducible transgenic line Tg (drl:hoxa9;hsp70:meis1) harboring hoxa9/meis1 under the draculin (drl) promoter. The downregulation of runx1, c-myb, mpx, mfap4, and gata1 in Tg (drl:hoxa9;hsp70:meis1) embryos indicated enforced hoxa9/meis1 perturbs embryonic hematopoiesis. Importantly, adult Tg (drl:hoxa9;hsp70:meis1) develops malignant myeloid disease with an abundance of myeloid precursor cells, anemia, and high mortality after a latency period (~5-months-aged) with comparable to murine model and human AML patients. Genome-wide transcription changes analysis indicated arrested differentiation genes such as gata2b, notch1b, and gfi1ab are upregulated. Leflunomide, inhibitor of enzyme dihydroorotate dehydrogenase (DHODH) which is a potential option for differentiation therapy of AML, relieves defective hematopoiesis in transgenic embryos and larvae. Collectively, we have identified an inducible malignant myeloid disease transgenic zebrafish model similar to AML and provided a unique opportunity for high-throughput in vivo chemical screening for AML therapy and study the related mechanisms.
Project description:The clustered homeobox proteins play crucial roles in development, hematopoiesis and leukemia yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 co-bind at hundreds of highly evolutionarily-conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation and transcriptional activation of a network of proto-oncogenes including Erg, Flt3, Lmo2, Myb and Sox4. Collectively this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia. Enriched Hoxa9 and Meis1 regions and a selected set of the nearest 360 TSSs were tiled onto Nimblegen custom arrays (Mus musculus 8, Feb 2006) with 50-mer probes (average spacing of 35bp), along with 60 negative control sequences that were selected randomly from the mouse genome