Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide ChIP-seq binding profiles and linked gene expression arrays

ABSTRACT: This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

ORGANISM(S): Mus musculus

SUBMITTER: Evangelia Diamanti

PROVIDER: E-GEOD-64069 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-scale definition of the transcriptional programme associated with compromised PU.1 activity in acute myeloid leukaemia.

Sive J I JI Basilico S S Hannah R R Kinston S J SJ Calero-Nieto F J FJ Göttgens B B

Leukemia 20150701 1

Transcriptional dysregulation is associated with haematological malignancy. Although mutations of the key haematopoietic transcription factor PU.1 are rare in human acute myeloid leukaemia (AML), they are common in murine models of radiation-induced AML, and PU.1 downregulation and/or dysfunction has been described in human AML patients carrying the fusion oncogenes RUNX1-ETO and PML-RARA. To study the transcriptional programmes associated with compromised PU.1 activity, we adapted a Pu.1-mutate ...[more]

PMID: 26126967

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Project description:Transcription factors are often regarded as having two distinct components: a DNA binding domain (DBD) to allow binding to the regulatory region of a target gene and a trans-acting functional domain (FD) to modulate gene expression. Recently, it is becoming clear that the DBDs of transcription factors alone are incapable of providing sufficient specificity to account for the highly complex genomic structures in eukaryotes. Other regions, outside of the DBD, may play a role in transcription factor DNA binding specificity in vivo. In order to test this hypothesis, we examined a model zinc finger transcription factor, KrÃ¼ppel-like factor 3 (KLF3) with a FD that recruits partner proteins for its repressive activity and a three zinc finger DBD. Previously, we showed that deletion of the entire KLF3 FD reduced DNA binding across the genome. This implied the importance of the FD for in vivo DNA binding specificity. In the current study, we fused the KLF3 FD onto an unrelated, but well characterised Artificial Zinc Finger (AZF) targeting a standard target gene, VEGF-A. We compared the genomic DNA binding profile of this chimeric KLF3 construct, termed KLF3FD-AZF, to that of the AZF alone. Chromatin Immunoprecipitation followed by next generation sequencing was used to assess genomic wide DNA-binding of these AZFs. Remarkably, in these gain-of-function experiments, we again observed that the KLF3 FD is critical for in vivo DNA binding specificity. The addition of KLF3 FD increased the number of binding sites by more than two fold compared to the AZF alone. KLF3 FD also directed more binding to the promoter regions. Further investigations of these acquired sites identified a substantial portion of these sites as the known endogenous KLF3 bound regions, whilst others contain sequences that are similar but not identical to the predicted AZF target sequence. These results clearly demonstrate that the specific localisation of this model transcription factor to target genes is not solely dependent on the DBD and that the regions outside of this domain may also play an important role. ChIP-Seq experiments were performed on four HEK293 cell lines, two each stably expressing AZF or KLF3FD.AZF or KLF3FD only (two biological replicates). Input samples were included as controls.

Project description:Purpose: The goals of this study are to assess the transcriptional networks governed by the transcription factor XBP1 in lineage-uncommitted myeloid progenitors and in eosinophil-committed myeloid progenitors. Methods: mRNA profiles of FACS-purified granulocyte-macrophage progenitors (GMPs) from XBP1 flox/flox or XBP1 flox/flox Vav1-Cre mice were generated by sequencing, in biological triplicates, using an Illumina HiSeq2000 sequencer. The Illumina HiSeq2000 sequencer was also used to obtain mRNA profiles of FACS-purified GMPs transduced with the transcription factor GATA2, resorted 36 hours post-transduction, and cultured for 48 hours, again in biological triplicates per genotype. Sequence data from Illumina's HiSeq2000 sequencer were demuxed to generate FASTQ files for each sample using Illumina's CASAVA pipeline (version 1.8.2). The reads that passed illumina's quality/purity filter were aligned to the mouse genome (Illumina iGenomes mm9 build) using STAR aligner (version 2.3.0) with default parameters. The resulting SAM alignment files were then converted to the BAM file format, sorted and indexed using SAMtools (version 0.1.14). Mapped reads were counted with the python module HTSeq, and differential expression analyzed with the Bioconductor package DESeq. Results and conclusions: By monitoring XBP1-dependent transcriptional changes at different stages of eosinophil development, we demonstrated that classical XBP1-dependent networks such as glycosylation, chaperone production, and ERAD were downregulated in GMPs prior to eosinophil commitment, though there were no major defects in differentiation or survival. However, mRNA profiling clearly demonstrated that XBP1 deficiency causes a state of cellular stress upon eosinophil commitment. The eosinophil transcriptome was largely intact, and most dysregulated genes were associated with ER stress. However key granule protein genes required for eosinophil development such as Prg2 and Epx were selectively downregulated only after eosinophil commitment, but not in pre-committed myeloid progenitors, and this correlated with Ingenuity Pathway Analysis predictions that GATA1 function was impaired. This study documents the interplay between cellular stress and the ability to maintain key facets of cellular differentiation. Analyses of XBP1-dependent transcriptional networks at two stages of eosinophil development.

Dataset Information

Genome-wide ChIP-seq binding profiles and linked gene expression arrays

Publications

Genome-scale definition of the transcriptional programme associated with compromised PU.1 activity in acute myeloid leukaemia.

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