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Updated: Jun 28, 2025
Proliferation and Differentiation of Murine Myeloid Precursor 32D/G-CSF-R Cells
Published on: February 21, 2018
PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human
Casey D S Katerndahl1, Olivia R S Rogers1, Ryan B Day1
1Division of Oncology, Department of Internal Medicine, Section of Stem Cell Biology, Washington University School of Medicine, St. Louis, MO 63110.
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 PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA 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 Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.
Insights
The PML::RARA fusion protein initiates acute promyelocytic leukemia by activating GATA2 expression. GATA2 binding creates accessible chromatin, enabling PML::RARA to bind DNA and drive disease progression.
Area of Science:
- Hematology
- Molecular Biology
- Cancer Biology
Background:
- The precise mechanism by which the PML::RARA fusion protein drives acute promyelocytic leukemia (APL) remains incompletely understood.
- Identifying the direct genomic targets and interacting partners of PML::RARA is crucial for elucidating APL pathogenesis.
Purpose of the Study:
- To define the genomic binding landscape of the PML::RARA fusion protein in hematopoietic progenitor cells.
- To investigate the role of chromatin accessibility and transcription factors, particularly GATA2, in PML::RARA-mediated leukemogenesis.
- To elucidate the functional relationship between PML::RARA and GATA2 in initiating the APL transcriptional program.
Main Methods:
- Chromatin immunoprecipitation sequencing (ChIP-seq) and CUT&RUN assays to map PML::RARA genomic binding sites.
- ATAC-seq to assess chromatin accessibility in hematopoietic progenitor cells.
- Proximity labeling and co-immunoprecipitation to identify PML::RARA interacting proteins.
- Gene manipulation (overexpression and inactivation) to study the functional role of GATA2.
Main Results:
- PML::RARA predominantly binds to pre-existing accessible chromatin regions in wild-type cells.
- GATA binding motifs and the pioneering factor GATA2 are significantly enriched near PML::RARA binding sites.
- PML::RARA interacts with approximately 250 proteins, including GATA2, with some interactions stabilized by DNA binding.
- GATA2 overexpression recapitulates key epigenetic and transcriptional changes induced by PML::RARA.
- Inactivation of GATA2 prior to PML::RARA expression blocks leukemogenesis and self-renewal induction.
Conclusions:
- PML::RARA initiates its oncogenic program by leveraging pre-existing accessible chromatin regions, often associated with GATA motifs.
- GATA2 acts as a critical mediator, potentially by creating accessible chromatin and interacting with PML::RARA.
- PML::RARA positively regulates GATA2 expression in a feed-forward loop, which is essential for establishing the leukemic transcriptional program and driving self-renewal.
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