PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

Abstract

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of <i>PML::RARA</i>, <i>Gata2</i> overexpression induces many of the same epigenetic and transcriptional changes as <i>PML::RARA</i>. These findings suggested that <i>PML::RARA</i> may indirectly initiate its transcriptional program by activating <i>Gata2</i> expression: Indeed, we demonstrated that inactivation of <i>Gata2</i> prior to <i>PML::RARA</i> expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating <i>Gata2</i> expression. <i>Gata2</i> may therefore be required for <i>PML::RARA</i> to establish its transcriptional program.