Proliferation-Independent Functions of the E2F-2 Transcription Factor during Terminal Erythroid Maturation

Lesions in the retinoblastoma (Rb) pathway induce defects in hematopoiesis that are typically thought to be due to inappropriate proliferative signaling from increased E2F transcriptional activity. Newer data demonstrate that E2F transcription factors have distinct roles in differentiating cells, but the key proliferation-independent functions of the E2Fs remain incompletely defined. We previously found that deregulated activity and expression of cyclin E induces defects in terminal erythroid cell maturation, using a mouse knock-in model (cyclin E^{T74A T393A}). Because cyclin E-Cdk2 potentiates E2F transcriptional activity via Rb phosphorylation, we hypothesized that inhibiting E2F activity would rescue hyperactive cyclin E-associated erythroid cell defects. E2F-2 is specifically induced during terminal erythroid maturation; therefore, we crossed our cyclin E knock-in strain with E2F-2 knockout mice. Unlike cyclin E knock-in bone marrows that display obvious defects in erythroid cell maturation, cyclin E knock-in; E2F-2 knockout animals demonstrated normalized erythroid maturation by flow cytometry. However, these compound mutant mice remain anemic, suggesting red blood cell (RBC) maturation was not completely restored.

We studied adult E2F-2 -/- mice further, and consistent with published data, we found that they are anemic. Interestingly, we do not detect obvious terminal erythroid maturation defects in adult E2F-2-knockout bone marrows. Using CFSE-labeled erythrocyte in vivo survival experiments, we found loss of E2F-2 results diminishes survival of adult peripheral erythroid cells within syngeneic, wild-type recipients, suggesting the anemia in the E2F-2 knockout mice is due to accelerated destruction and not solely a production defect. To study the erythroid maturation program in the absence of E2F-2 in detail, we obtained fetal liver-derived hematopoietic progenitors from E2F-2 knockout versus wild-type embryos and differentiated these towards the erythroid lineage in vitro. E2F-2 deletion results in impaired erythroid maturation as evidenced by flow cytometry-based assays of cell surface marker expression, abnormal cell morphologies, and impaired enucleation. In order to identify functionally significant E2F-2 targets during erythroid maturation, we performed microarray analyses on sorted subpopulations of fetal liver-derived erythroid cells. We found widespread gene expression changes in E2F-2 knockout cells, which included but were not limited to proliferation related pathways. Furthermore, using global histone mass spectrometry analysis, we found that loss of E2F-2 results in marked alteration of histone-H3, lysine-4 methylation during erythroid differentiation. Thus, our data demonstrate that E2F-2 has both proliferation-dependent and independent functions that include the coordination of transcriptional and epigenetic programs during terminal erythroid maturation.