Current work indicates that transcriptional repression is at least as important as transcriptional activation in normal development. Inappropriate or untimely transcriptional repression in immature hematopoietic cells is often the basis for a block to differentiation in hematologic malignancies. Activation of PU.1, a myeloid and B-cell specific transcription factor, in erythroid cells plays a key role in Friend virus-induced mouse erythroleukemia (MEL). Previous results from our laboratory showed that PU.1 blocks the erythroid differentiation-promoting activity of GATA-1 by binding directly to GATA-1 on DNA and inhibiting its transcriptional function. PU.1-mediated repression of GATA-1 on transiently transfected GATA-1 target genes is dependent on the corepressor pRb that also binds to PU.1 (Rekhtman et al., Genes & Dev 1999 and Mol Cell Biol 2003). To further investigate the mechanism of PU.1-mediated repression of GATA-1 in chromatin, we examined the occupancy of several GATA-1 target genes by PU.1 and pRb, as well as the state of core histone modifications at these loci in MEL cells by quantitative chromatin immunoprecipitation. These studies included both endogenous GATA-1 target genes and an exogenous GATA-1 target gene (alpha globin) integrated at a specific locus in MEL cells by Recombinase-Mediated Cassette Exchange. We found that GATA-1 sites at both the exogenous, integrated gene as well as at endogenous genes (including the regulatory regions of the alpha globin, beta globin, alas-e, eklf, p45 nf-e2) are occupied by a GATA1 - PU.1 - pRb complex in undifferentiated MEL cells. The presence of all three components of the complex is dependent on intact GATA-1 binding sites in the exogenous, integrated gene. The histone methyltransferase Suv39H1 and the histone H3MeK9 binding protein, HP1alpha, are also present at the repressed loci. During induced differentiation of MEL cells, PU.1, pRb, Suv39H1 and HP1alpha occupancy at these sites declines but GATA-1 continues to be present at its binding sites. The disruption of the repression complex at these loci during differentiation as well as during siRNA-mediated PU.1 knock down is associated with conversion of methylated H3K9 to acetylated H3K9 and significant transcriptional derepression of these GATA-1 target genes. These findings support a model for repression of GATA-1 by PU.1 at endogenous loci through recruitment of the corepressor pRb and associated histone methyltransferase (Suv39H1) and H3MeK9 binding (HP1alpha) activities.