Definition of a Small Core Transcriptional Circuit Regulated by AML1-ETO [PRO-seq]

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. Utilizing CRISPR-based genome editing, we inserted a degron tag into the endogenous AML1-ETO locus of Kasumi-1 cells, as well as overexpressed a degradable AML1-ETO protein in CD34+ human cord blood cells. This allowed rapid AML1-ETO protein degradation upon addition of a proteolysis targeting chimera (PROTAC). Furthermore, by combining rapid degradation with nascent transcript analysis (PRO-seq), RNA-seq and Cut&Run, we have defined the core AML1-ETO regulatory network. De-repression of this small set of genes set off a cascade of transcriptional events resulting in myeloid differentiation. PRO-seq was performed on biological replicates. Parental Kasumi-1 cells (wild-type AML1-ETO) or untreated Kasumi-1 cells expressing AML1-ETO-FKBP12F36V served as negative controls. Cells were harvested at 30 min, 1 hr, 2 hr, 6 hr and 24 hr post-treatment with 500 nM dTAG-47, as well as at 24 hours following treatment with 100 nM GSK2879552 or 500nM dTAG-47 + 100 nM GSK2879552. Nuclei were isolated from cells for nuclear run-ons.