Abstract
Hematopoiesis is the process by which the blood of the body is generated. This is a tightly regulated process with many players involved to carry out high fidelity proliferation and differentiation of stem cells into specialized cells. These players are transcription factors. Transcription factors are regulated to be present in the correct quantity, at the correct time and place during stem cell differentiation in order to create the necessary cells required by the body. However, in some cases, this regulation is disconcerted. This misregulation gives rise to a disease, leukemia, that is characterized by aberrant proliferation and stem cell-like states. Our work will explore a transcription factor, AF9, that shows a previously undiscovered RNA binding attribute that is required for hematopoiesis. We will also describe efforts to target a transcription factor, ERG, whose aberrant activity is implicated in multiple leukemias.
The protein AF9 (MLLT3) plays a critical role in hematopoiesis. Increased expression of AF9 is observed in the megakaryocyte and erythrocyte hematopoietic lineage. The mechanism by which hematopoietic stem cells are driven toward this lineage is still not completely understood, but upregulation of AF9 gene targets, such as GATA2, lead to this lineage determination. AF9 has a well-documented role in the Super Elongation Complex and in DOT1L-mediated methylation of H3K79. These two functions are important for gene activation and allow for rapid upregulation of AF9 targets. Recently, work has shown the AF9 YEATS domain is a reader of histone acetylation and crotonylation marks, thus linking H3K79 methylation and transcriptional elongation to histone acetylation activation marks. The YEATS domain is the first discovered reader of histone crotonyl marks.
We present here data that shows that the AF9 YEATS domain is also able to bind to RNA. Using a PAR-CLIP based approach we were able to show that AF9 RNA binding is relevant in a cellular context by demonstrating that AF9 co-precipitates with RNA in a functionally significant cell line. Ten putative binding targets of the AF9-RNA interaction were identified. Finally, we show that AF9 RNA binding is functionally important for hematopoiesis by engineering an AF9 mutant, K67E, which abrogated RNA binding without impacting AF9’s other functions. Using this mutant we were able to show that selectively inhibiting AF9 RNA binding alters differentiation and gene expression in mouse bone marrow cells in a manner that nearly completely mimics a complete AF9 knockout ex vivo.
The protein ERG has been shown to play an essential role in hematopoiesis. Knockouts are unable to properly populate the blood compartment. Translocation of ERG has been observed in multiple leukemias. In these instances, ERG expression is controlled by a promoter element that aberrantly upregulates expression of ERG in leukemic cells. Indeed, even in leukemic cells that do not possess a mutated ERG gene, expression of ERG is correlated with a more aggressive cancer that is prone to relapse.
We present data here that shows a means of specifically targeting ERG DNA binding, and thereby transcriptional activity. This targeting takes advantage of a unique autoinhibition process that ERG undergoes. This process is unique to ERG and provides a means of defining a small molecule specific to ERG DNA binding activity with no off-target effects on other proteins that contain the homologous Ets domain. We are able to show that our small molecule inhibitor of DNA binding binds directly to ERG to accomplish said activity in an allosteric manner. Furthermore, we have data to show that this inhibition is accomplished by stabilization of autoinhibitory domains to shift the autoinhibition equilibrium towards an inactive state.
