1020 – TARGETED PROTEIN DEGRADATION DEFINES DIRECT TARGETS AND MECHANISMS OF CONTROL BY RUNX1/ETO

Abstract

Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements. Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements.