PU.1 and pRB Interact and Cooperate To Repress GATA-1 and Block Erythroid Differentiation

Abstract

PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each other's function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.

The role of Ikaros in human erythroid differentiation

“…13 PU1-GATA1 antagonism is a well documented mechanism in myeloid versus erythroid differentiation. 45 However, our preliminary results suggest that Ikaros does not significantly affect human myeloid differentiation.…”

Critical Roles for PU.1, GATA1, and GATA2 in the Expression of Human FcεRI on Mast Cells: PU.1 and GATA1 Transactivate FCER1A, and GATA2 Transactivates FCER1A and MS4A2

“…(GATA) inhibits the function of GATA (PU.1), resulting in dominion by itself, during the development from hematopoietic stem cells toward the monocyte (erythrocyte/megakaryocyte) lineage (21)(22)(23)(24)(25)(26)(27)(28). In contrast, PU.1 and GATA1/GATA2 exhibit a cooperative function in mast cells, as follows: 1) PU.1 and GATA2 cooperatively induce mast cell development (20); 2) PU.1 and GATA1 synergistically transactivate cell type-specific gene regulation in mast cells (5,29); and 3) PU.1 is required for GATA1 expression in mast cells (30).…”

The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis

“…The mechanism of such stoichiometry can operate in the absence of DNA-binding sites and therefore the transcription factors can either mutually block each other's accession to DNA 56 or can coexist on DNA and block each other's transactivation by protein-protein interactions within chromatin. 49,57 Initially it was thought that PU.1 prevents binding of GATA-1 to DNA as shown by electrophoretic mobility shift assays. 56 However, by using DNase-I footprinting such inhibition was observed at a non-physiological molar ratio (20:1) whereas at a 1:1 ratio PU.1 actually altered the GATA-1-generated footprint near the GATA-1-binding site thereby arguing for the possibility that both PU.1 and GATA-1 coexists on DNA.…”

“…56 However, by using DNase-I footprinting such inhibition was observed at a non-physiological molar ratio (20:1) whereas at a 1:1 ratio PU.1 actually altered the GATA-1-generated footprint near the GATA-1-binding site thereby arguing for the possibility that both PU.1 and GATA-1 coexists on DNA. 57 The GATA-1 reporter construct was transfected into U2OS cells, a human osteosarcoma cell line, and in this in vivo assay it clearly responded positively to GATA-1 and negatively following the addition of PU.1. 57 Chromatin immunoprecipitation studies showed the co-occupancy of both PU.1 and GATA-1 proteins to DNA 57 and that this co-occupancy was dependent on the presence of both a GATA-1 cis-binding site and the Ets domain on the PU.1 protein.…”

“…57 The GATA-1 reporter construct was transfected into U2OS cells, a human osteosarcoma cell line, and in this in vivo assay it clearly responded positively to GATA-1 and negatively following the addition of PU.1. 57 Chromatin immunoprecipitation studies showed the co-occupancy of both PU.1 and GATA-1 proteins to DNA 57 and that this co-occupancy was dependent on the presence of both a GATA-1 cis-binding site and the Ets domain on the PU.1 protein. Interestingly, the N-terminal portion of PU.1 does not affect the occupancy of PU.1 and GATA-1 on its DNA-binding site 57 yet is required to bind with the retinoblastoma protein (pRb) for repression of GATA-1.…”