ABSTRACT: Here we investigated the effects of CEBPA transcription factor expression on myeloid NB4 cells. The sequence of rat CEBPA was C-terminally fused to a promiscuous biotin ligase tag (BirA*) and NB4 cell lines were engineered to express the fusion protein under the control of a doxycycline inducible promoter. Three different NB4 cell lines were investigated that expressed (i) BirA* tag alone (ii) full length CEBPA isoform (P42) fused to BirA* (iii) truncated CEBPA isoform (P30) fused to BirA*. Cells were seeded in media supplemented with or without doxycycline.
Project description:Treatment of acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA) results in terminal differentiation of leukemic cells toward neutrophil granulocytes. Administration of ATRA leads to massive changes in gene expression, including down-regulation of cell proliferation-related genes and induction of genes involved in immune function. One of the most induced genes in APL NB4 cells is transglutaminase 2 (TG2). RNAi-mediated stable silencing of TG2 in NB4 cells (TG2-KD NB4) coupled with whole genome microarray analysis revealed that TG2 is involved in the expression of a large number of ATRA-regulated genes. The affected genes participate in granulocyte functions and their silencing lead to reduced adhesive, migratory and phagocytic capacity of neutrophils and less superoxide production. The expression of genes related to cell cycle control also changed, suggesting that TG2 regulates myeloid cell differentiation. CC chemokines CCL2, 3, 22, 24 and cytokines IL1B and IL8 involved in the development of differentiation syndrome (DS) are expressed at significantly lower levels in TG2-KD NB4 cells than in wild-type NB4 cells upon ATRA treatment. Based on our results, we propose that reduced expression of TG2 in differentiating APL cells may suppress effector functions of neutrophil granulocytes and attenuate the ATRA-induced inflammatory phenotype of DS. We used microarrays to detail the global program of gene expression underlying ATRA-induced differentiation of TG2 knockout NB4 cells. TG2 knockout NB4 cells were differentiated for 48 and 72 hours in the presence of ATRA and their gene expression profiles were compared to the wild-type cells at the same time points. Undifferentiated wild-type and TG2 knockout NB4 cells were used as untreated controls. Three biological replicates each.
Project description:[1] Gene expression profiling in Gata4, Mef2a knockdown in HL-1 cells. HL-1 cells infected with adenovirus expressing either control siRNA or Gata4, and Gata4 & Mef2a siRNA. [2] Genome-wide maps of cardiac transcription factors binding region in HL-1 cells. We performed bio-ChIP-seq using streptavidin beads immunoprecipitation of biotinylated 5 cardiac transcription factors (fbio) and P300 antibody ChIP-seq. We used a dox-inducible dual adenovirus system to express biotinylated Core Cardiac TFs in HL1. One adenovirus expressed the dox-activated reverse tet activator protein (rtTA) and the E. coli biotinylating enzyme BirA from the cardiac specific rat troponin T promoter. The second virus expressed a Core Cardiac TF fused to a C-terminal flag-bio epitope tag, where bio is the 15 amino acid substrate for BirA. This in vivo biotinylation approach permits pulldown of different factors to be performed under identical, stringent conditions, and circumvents limitations posed by available antibodies. We titrated adenovirus and dox doses to express GATA4flbio and MEF2Aflbio at near endogenous levels. The same conditions were then used to express SRFflbio, TBX5flbio, and NKX2-5flbio. Reference: 12. de Boer E, Rodriguez P, Bonte E, Krijgsveld J, Katsantoni E et al. (2003) Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc Natl Acad Sci U S A 100: 7480-7485. [1] Gene expression profiling: Identify differentially expressed genes after siRNA Gata4 or Gata4_Mef2a double knockdown in HL-1 cells [2] Genome occupancy profiling: Identify cardiac active enhancers by identified 5 cardiac transcription factors and P300 peaks.
Project description:NB4 is an APL derived cell line, carrying the t(15;17) translocation and expressing the PML/RARa fusion protein. Still, an important question that remains to be addressed is whether PML/RARa target genes are transcriptionally suppressed in primary APL cells and re-activated in all-trans retinoic acid (ATRA) treated NB4 cells. Gene expression of NB4 cells treated with ATRA at different time points were analyzed. Experiment Overall Design: 6 samples at various times. No replicats.
Project description:To better understand the mechanisms of blockage of myeloid differentiation and apoptosis and induction of proliferation by miR-125b, we proceeded to identify miR-125b target genes involved in these pathways. We analyzed the total cellular gene expression pattern by RNA-sequencing of the parental NB4 myeloid cell line and that transiently transfected with miR-125b. We generated four cDNA libraries corresponding to duplicates of miR-125b and control cells. Compare the gene expression levels in miR control transfected cells with that in miR-125b transfected NB4 cells.
Project description:Chromatin accessibility is a key determinant of cell-type-specific gene expression. Here, we have investigated the chromatin architecture of different acute myeloid leukemia (AML) cells and the changes in accessibility when NB4 (APL) cells undergo the process of differentiation. For nuclease-accessible site sequencing (NA-seq; Gargiulo et al. 2009), chromatin-accessible libraries were generated in different AML leukemic cells by using restriction enzymes NlaIII and HpaII. In the case of NB4 cells, accessibility was mapped both before and after treatment with all-trans retinoic acid (ATRA) for 48hr. Differences were observed between the two conditions, and chromatin accessibility was correlated with underlying epigenetic modifications. For validation purposes, NA-seq libraries (using the NlaIII enzyme) were generated in APL and AML M1 patient's blasts. All of the ChIP-seq (Martens et al. 2010) studies were performed in leukemic NB4 and SKNO-1 cells. Supplementary file 'GSE30254_All_accessibleregions_ATRA_NB4_fseq.wig' includes data for Samples GSM749512, GSM749513, GSM749516, and GSM749517. Supplementary file 'GSE30254_All_accessibleregions_untreated_NB4_fseq.wig' includes data for Samples GSM749510, GSM749511, GSM749514, and GSM749515.
Project description:ATRA was identified as a Pin1 inhibitor via fluorescence polarization-based high throughput screening. We performed microarray expression profiling to demonstrate the similarity between ATRA and Pin1 KD at the genome-wide level APL NB4 cells in response to ATRRA or inducible Pin1 knockdown for 3 days were collected for RNA extraction and hybridization on Affymetrix microarrays. We sought to validate in genome-wide level whether similarity occurred between ATRA and Pin1 knockdown-treated NB4 cells.
Project description:Overexpression of antiapoptotic BCL2 family proteins occurs in various hematologic malignanices and contributes to leukemogenesis by inhibiting the apoptotic machinery of the cells. BH3 mimetics provide an option for medication, with venetoclax as the first drug applied for chronic lymphocytic leukemia and for acute myeloid leukemia. To find additional hematologic entities with ectopic expression of BCL2 family members, we performed expression screening applying the LL-100 panel. Primary effusion lymphoma (PEL) and anaplastic large cell lymphoma (ALCL), 2/22 entities covered by this panel, stood out by high expression of MCL1 and low expression of BCL2. The MCL1 inhibitor AZD-5991 induced apoptosis in both malignancies suggesting that this BH3 mimetic might be efficient as drug for these diseases. The ALCL cell lines also expressed BCLXL and BCL2A1, both contributing to survival of the cells. The combination of specific BH3 mimetics yielded synergistic effects, pointing to a novel strategy for the treatment of ALCL. The PI3K/AKT inhibitor BEZ-235 could also efficiently be applied in combination with AZD-5991, providing an alternative to avoid thrombocytothemia, which is associated with the use of BCLXL inhibitors.
Project description:We report the genomic localization of cohesin oligomers in nocodazole arrested yeast cells. Two alleles of SMC3 were expressed in yeast cells, one fused to BirA enzyme and the other tagged with AviTag. Cohesin oligomers were biotinylated and ChIP with streptavidin beads. As control experiments, cohesin localization on chromosome was determined in strains expresses freely diffusable BirA enzyme, where all Smc3 proteins were biotinylated; non-specific ChIP were determined in strains with no BirA.
Project description:Purpose of these experiments was to assess the effect of glucocorticoid receptor SUMOylation on its protein-protein interactions using BioID. Experiments were performed with SUMOylation deficient GR3KR (K277,293,703R) fused to the BirA* biotin ligase tag.