Amplification of pico-scale DNA mediated by bacterial carrier DNA for small-cell-number transcription factor ChIP-seq
ABSTRACT: ChIP-seq is used to map transcription factor occupancy and generate epigenetic profiles genome-wide. The requirement of nano-scale ChIP DNA for generation of sequencing libraries has impeded ChIP-seq on in vivo tissues of low cell numbers. We describe a robust, simple and scalable methodology for ChIP-seq of low-abundant cell populations, reliably amplifying 50 pg of ChIP DNA, corresponding to ~30,000 input cells for transcription factor ChIP (CEBPA) and 3,000 cells for histone mark ChIP (H3K27me3). This represents a significant advance compared to existing technologies, which involve complex and time-consuming steps of pre-amplification, making them susceptible to experimental biases. ChIP-seq of histone modifications H3K27me3 (2 biological replicates (I+II) , 2 ng input), H3K4me3 (2 biological replicates (II+III), 2 ng input), transcription factor CEBPA (2 biological replicates (I+II), 300 pg input), 4 diluted CEBPA libraries (pool of ChIP from 3 biol. replicates (I+II+III) 3x 100 pg input, 1x 50 pg). Additonal ChIP-seq using 10,000 cells, 1 biological replicate of each H3K4me3 and CEBPA.
Project description:This study aims to investigate whether the passage of human chromosome 21 through the mouse male germline results in changes in the transcriptional deployment of the exogenous chromosome in the offspring generation. We used the Tc1 mouse model that stably carries almost an entire copy of human chromosome 21 and profiled the genome-wide pattern of H3K4me3, H3K27ac, CEBPA, HNF4A and RNA polymerase II in liver tissue of male and female-germline derived Tc1 mice using ChIP-Seq. Furthermore, the genome-wide pattern of H3K4me3 was profiled in additional tissues including kidney, liver and brain.
Project description:We profiled the genome-wide occupancy of three tissue-specific transcription factors, HNF4A, CEBPA and FOXA1, as well as the genome-wide occurrence of the histone mark, H3K4me3 in the livers of two inbred parental mouse strains (C57BL/6J and CAST/EiJ) and their F1 crosses. We also included H3K27ac data generated from F1 hybrids as well as the profiling of HNF4A, CEBPA and FOXA1 in both CEBPA and HNF4a heterozygous knock-outs.
Project description:Exogenous overexpression of CEBPA transcription factor induces transdifferentiation from pro B cells into functional macrophages. Here we report the CEBPA binding ChIP-Seq data at 12 hours time point after induction of transdifferentiation in human BLaER1 cells (Rapino et al. 2013).
Project description:The key myeloid transcription factor (TF) CEBPA is frequently mutated in acute myeloid leukemia (AML), but the molecular ramifications of this leukemic driver mutation remain elusive. To investigate CEBPA mutant AML, we compared gene expression changes in human CEBPA mutant AML and in the corresponding CebpaLp30 mouse model, and identified a conserved cross-species transcriptional program. ChIP-seq revealed aberrantly activated enhancers, exclusively occupied by the leukemia-associated CEBPA-p30 isoform. One leukemic-enhancer upstream of Nt5e, encoding CD73, was physically and functionally linked to this conserved AML gene, and could be activated by CEBPA. Targeting of CD73-adenosine signaling increased AML survival in transplanted mice. Our data indicate a first-in-class link between a TF cancer driver mutation and a druggable, direct transcriptional target.
Project description:Canton S flies (as wild type) were bred at 25C and tightly staged 2-hr embryos were collected after 2 times of 2-hr prelay. The embryos were aged on the apple juice plates and aged to 4-6 and 6-8 hr. For each time point, two independent Chromatin immunoprecipitation (ChIP) experiments were performed using two different antibodies and compared to two independent mock ChIPs using the corresponding pre-immune serum. One Zfh1 antibody was generated in Guinea pig against full-length Zfh1 in the Furlong laboratory, and the other was a generous gift from Dr. Ruth Lehmann. ChIPs were optimized using the enrichment of a binding site identified in a pilot experiment of Zfh1- ChIP-on-chip measured by real-time PCR as previously described. The quality of each IP was also assessed by real-time PCR. Solexa libraries were prepared according to the manufacturer's recommendations. Library quality was assessed on a 2100 Bioanalyzer system (Agilent). All samples were single-end sequenced with 36-bp reads using an Illumina Genome Analyzer IIX by the EMBL Genomics Core Facility.
Project description:This study aims to study genome wide location of HNF4a, FoxA1 and CEBPA in wild type rat liver. Please note that all raw data files for this study were replaced with new versions on 12 March 2014 because the previous versions are corrupted. The corrupted files are associated with ENA run accessions ERR215698 to ERR215705 and should not be used. The correct files are associated with ENA run accessions ERR458085 to ERR458092.
Project description:Expression analysis of CEBPa knockout effects on gene expression in adult mouse liver. We used a conditional knock out strategy to delete CEBPa specifically in adult mouse hepatocytes and analysed the resulting changes in gene expression by means of microarrays.
Project description:Analysis of global RNA expression of Cebpa knockdown lineage-negative marrow cells reveals known and potentially novel C/EBPa targets. Total RNA from lineage-negative marrow progenitors transduced with vector or Cebpa shRNA and cultured under myeloid differentiation condition for 2 days were compared.
Project description:Purpose: CEBPA mutations are found as either biallelic (biCEBPA) or monoallelic (moCEBPA). We set out to explore whether the kind of CEBPA mutation is of prognostic relevance in cytogenetically normal AML (CN-AML). Patients and Methods: 467 homogeneously treated CN-AML patients were subdivided into moCEBPA, biCEBPA and wildtype (wt) CEBPA patients. The subgroups were analyzed for clinical parameters and for additional mutations in the NPM1, FLT3 and MLL genes. Furthermore, we obtained gene expression profiles (GEP) for a subgroup of 61 patients using oligonucleotide microarrays. 61 bone-marrow samples from CN-AML patients were analyzed using Affymetrix HG-U133 oligonucleotide microarrays (Affymetrix, Santa Clara, CA). Sample preparation, hybridization and image acquisition were performed according to standard Affymetrix protocols. Custom chip definition files based on the GeneAnnot database were used for data analysis (Ferrari et al, BMC Bioinformatics 8:446). The twilight algorithm was used to comapre gene expression profiles of patients with wildtype CEBPA and mono- and biallelic CEBPA mutations.