Project description:Acute myeloid leukemia (AML) is caused by recurrent mutations in members of the gene regulatory and signalling machinery subverting hematopoietic differentiation. We previously showed that each AML sub-type displays a different gene regulatory network with specific transcription factor families playing distinct roles in maintaining the leukemic phenotype. Here we show that the transcription factor WT1 forms a major node in the gene regulatory networks of multiple AML sub-types. It is frequently mutated and up-regulated in AML and its expression is pedictive for relapse. The WT1 protein exists as different isoforms which we show here to harbor differential chromatin binding and contrasting biological activity, including directing differential splicing. For two main AML subtypes we demonstrate that WT1 responds to oncogenic signaling and is part of a signaling-responsive transcription factor hub that controls AML growth. WT1 therefore plays a central and wide-spread role in AML biology.
Project description:DNase hypersensitivity using Nimblegen ENCODE arrays (DNase-chip) in primary human trachea epithelia (pHTE), normal human bronchial epithelia (NHBE), Caco2, HT29, human skin fibroblasts, primary human male epididymis. DNase hypersensitivity mapping is used to detect putative regulatory elements of the human genome. We digested chromatin from the above cell types with DNaseI using the DNase-chip protocol devised by Crawford et al. (Nat. Methods, 2006). Briefly, cells are lysed, chromatin is digested with increasing amounts of DNase I, digested ends are blunted in adequetely digested samples, ligated to biotinylated linkers, purified with streptavidin beads, amplified by LM-PCR. As control, randomly sonicated genomic DNA is used. LM-PCR material was labeled and hybridized to hg_17 ENCODE arrays at Nimblegen facility. DNaseI-digested chromatin was hybridized to Nimblegen ENCODE arrays (build hg17). Randomly sonicated genomic DNA was used as control. Three biological replicates on three arrays were performed for Caco2 cells, three replicates were done with skin fibroblasts (two digestions were pooled and hybridized to a single array [Sample: SkinFibro.sample2]), two replicates on two arrays were performed for pHTE, NHBE, and HT29, and a single experiment for primary male epididymis.
Project description:DNase hypersensitivity using Nimblegen ENCODE arrays (DNase-chip) in primary human trachea epithelia (pHTE), normal human bronchial epithelia (NHBE), Caco2, HT29, human skin fibroblasts, primary human male epididymis. DNase hypersensitivity mapping is used to detect putative regulatory elements of the human genome. We digested chromatin from the above cell types with DNaseI using the DNase-chip protocol devised by Crawford et al. (Nat. Methods, 2006). Briefly, cells are lysed, chromatin is digested with increasing amounts of DNase I, digested ends are blunted in adequetely digested samples, ligated to biotinylated linkers, purified with streptavidin beads, amplified by LM-PCR. As control, randomly sonicated genomic DNA is used. LM-PCR material was labeled and hybridized to hg_17 ENCODE arrays at Nimblegen facility.
Project description:The Wilms' tumor suppressor gene (WT1) encodes a zinc finger transcription factor that plays important roles during development of several organs including metanephric kidneys. A number of WT1 target genes have been identified, but the detailed mechanisms by which WT1 orchestrates renal development remain elusive. To identify WT1 target genes relevant to development, genome-wide expression profiling was performed using oligonucleotide microarrays representing 39,000 human transcripts Keywords: Wilms' tumor 1 (WT1) gene, -KTS isoform; time course
Project description:We report the high-throughput profiling of histone modification and DNase I hypersensitivity sites in prostate cancer and breaset cancer cells. We found that while AR binding is associated with nucleosome depletion, ER binding is not. We showed that a quantitative measure of DNase I hypersensitivity changes is a powerful tool in indentifying transcription factor cistromes. Examination of histone modification marked nucleosomes and Dnase I hypersensitivity in prostate cancer and breast cancer cells with and without hormone treatment.