Genome binding/occupancy profiling by high throughput sequencing
Summary
Chromatin modifications are thought to provide additional context dependence for sequence-based gene activation. Binding sites of the transcription factor (TF) and important tumor suppressor p53 are unusually diverse with regards to their chromatin accessibility and histone modifications, suggesting different modes of binding. Here, we show that the ability of p53 to open chromatin and activate its target genes upon DNA damage is locally restricted by its cofactor Trim24. The histone-binding domains of Trim24 limit the role of p53 at closed chromatin but not at accessible chromatin where Trim24 is blocked by histone 3 methylation at lysine 4. In turn p53 regulates gene expression as a function of the naïve chromatin state prior to activation. These findings establish a set of p53 response genes in closed chromatin that are Trim24-regulated and illustrate how histone modification sensing cofactors bridge local chromatin state and TF potency.
Overall design
Examination of p53 and Trim24 function in cell lines including mouse embryonic stem cells. Includes ATACseq in WT mouse ESCs in untreated/p53activated conditions (duplicates), ATACseq in p53-degron tagged mouse ESC lines under p53degraded/untreated/p53activated conditions (duplicates), ATACseq in Trim24-degron tagged mouse ESC lines under Trim24degraded/untreated/p53activated/p53activated;Trim24degraded conditions (duplicates), ATACseq in Trim24-degron tagged mouse ESC line under p53activated/p53activated;Trim24degraded conditions at multiple timepoints after treatment including 0 hours/1 hours (duplicates)/2 hours (duplicates)/4 hours (duplicates)/8 hours (duplicates)/12 hours, ATACseq in Trim24-degron tagged mouse ESC line under p53activated/p53activated;Trim24degraded conditions with alternative p53 activation conditions by nutlin3a (20uM) or irradiation (60Gy)(duplicates). All conditions to activate p53 or degrade degron tagged alleles performed for 4 hours unless specified in a timecourse series.
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