Identification of zfp148 (ZBP-89) as a Novel GATA-1 Associated Transcription Factor Involved in Megakaryopoiesis and Definitive Erythropoiesis.

The zinc finger transcription factor GATA-1 plays an essential role in megakaryocytic and erythroid terminal maturation. Yet the molecular mechanisms that control GATA-1 function are incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. Here, we show that GATA-1 participates in stable multiprotein complexes ranging from ~100 kDa to greater than 600 kDa in murine L8057 megakaryoblastic cells. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and performed tandem anti-FLAG immunoaffinity and streptavidin affinity purification of GATA-1 associated complexes. Mass spectrometry (LC/MS/MS) of co-purified proteins identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β, SP1 and all components of the NuRD complex. In addition, we reproducibly co-purified zfp148 (also called ZBP-89/BERF-1/BFCOL-1, mtβ), a ubiquitously expressed Kruppel-type zinc finger transcription factor that binds GC-rich DNA elements. Zfp148 also co-purified with GATA-1 in erythroid (MEL) cells. Physical interaction between GATA-1 and zfp148 was confirmed by co-immunoprecipitation experiments using an anti-zfp148 antibody, and their interaction domains mapped to a C-terminal region of zfp148 and the zinc finger domain of GATA-1. Morpholino-mediated knock-down of zfp148 expression in zebrafish embryos ablates thrombocyte (equivalent to mammalian megakaryocyte/platelet) development, but does not significantly affect erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development). In vitro differentiation of murine ES cells containing homozygous disruption zfp148 by genetrap insertion (zfp148^gt/gt) shows essentially normal primitive erythropoiesis, but markedly impaired megakaryocytic and definitive erythropoiesis. Preliminary chimeric studies in adult mice demonstrate a failure of zfp148^gt/gt ES cells to contribute to mature erythrocytes. These findings suggest that zfp148 cooperates with GATA-1 to control differentiation of erythroid and megakaryocytic lineages. The differential requirement of zfp148 for definitive, but not primitive, erythropoiesis raises the possibility of a role in developmental hemoglobin switching.