PU.1 Relieves Its GATA-1-Mediated Repression near Cebpa and Cbfb During Transdifferentiation of Murine Erythroleukemia - Tool of Inducing Leukemic Blasts to Differentiate.

Abstract 547

Transcription factors GATA-1 and PU.1 interact on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that co-express GATA-1 and PU.1 are blocked at the blast stage but respond to down-regulation of PU.1 or up-regulation of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode two key hematopoietic transcription factors. Inhibition of GATA-1 by siRNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb co-occupied by PU.1 and GATA-1 in the leukemic blasts. Substantial derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we demonstrate that either activation of PU.1 or inhibition of GATA-1 efficiently reverse the transcriptional block imposed by GATA-1 and lead to activation of a myeloid transcriptional program directed by PU.1.

The mechanism of PU.1 and GATA-1 in leukemic state and upon leukemia differentiation involves the following putative steps: at myeloid genes such as Cebpa, PU.1 binds directly to DNA but is repressed by GATA1 that binds directly to PU.1 molecules on DNA. Activation of PU.1-ER and stable levels of GATA-1 create excess of availabel PU.1, which is not paired by availabel GATA-1 on DNA, allowing thus gene activation. Similarly, on erythroid genes such as Nfe2, GATA1 is bound to DNA, but is repressed by PU.1 that binds to this GATA-1 molecule. Activation of GATA1-ER creates an excess of availabel GATA-1 which is not paired on DNA by availabel PU.1, also allowing gene activation. Our mechanistic study implicates that transcription factor manipulation, such as inhibition of GATA-1 or activation of PU.1 in erythroleukemias, may represent an efficient tool of inducing leukemic blasts to differentiate. (Grants NR9021-4, 10310-3, 2B06077)