Identification of targetted gene loci by MIXL1 in the AML cell line U937

While the role of the paired-type homeobox transcription factor MIXL1 in early ebryonic development has been previously established, very little is known about the role of MIXL1 in adult hematopoiesis. To determine what role MIXL1 plays in adult hematopoietic cells, we used an established enforced expression line for FLAG-tagged MIXL1 in U937, then isolated targetted regions of the genome by ChIP-Seq. Through this analysis, we identified new putative direct transcirptional targets for MIXL1 in AML and hematopoiesis. Overall design: In attempt to identify the downstream role of MIXL1 in adult hematopoietic cells, we used two previously established MIXL1 overexpression lines (1MIXL and 2MIXL) and empty vector control (Control) clone of the AML cell line U937. 90% RPMI/10% FBS for 48 hours at a starting concentration of 1x105/ml before harvestings. Protein-DNA complexes were isolated from sonicated, crosslinked whole cell lysates using IgG mouse (IgG Control) or Flag antibodies. The purified DNA from these complexes for each sample was sequenced and analyzed for ChIP-Seq, then experimental samples (Flag-IP 1MIXL and 2MIXL) were normalized to control samples (Flag-IP or IgG-IP Control) to determine which regions of the genome MIXL1 interacted with.