Identification Of Pu.1 Target Genes That Are Dependent On Specific Functional Domains Of The Transcription Factor Pu.1.

The expression of PU.1, a member of the ets transcription factor family, is limited to the hematopoietic lineage. Using knockout and reduced PU.1 expression and gain of function mice as a model system, it has been demonstrated that PU.1 plays a key role in early myeloid and lymphoid fate decision and at later stages of myeloid differentiation and function. In PU.1 ( Sfpi1 ) null mice, dendritic cells and monocytes could not be identified, but early myeloid progenitors and immature neutrophils were present. In mice with reduced PU.1 expression monocyte development was hindered, immature neutrophil development ensued, and the mice developed acute myeloid leukemia. The different domains of the PU.1 transcription factors have been attributed to distinct functions in early myeloid fate decision. The PU.1 transcription factor is composed of 3 major domains: transactivation, PEST, and DNA binding. We have used an immature myeloid cell line (503) derived from the Sfpi1 -null mouse to analyze the effect of lentiviral gene delivery of PU.1 mutants lacking specific PU.1 domains, on differentiation of 503 cells, and to identify PU.1 target genes that are dependent on these specific domains for their expression. We found that a PU.1 mutant lacking the acidic domain leads to differentiation of the 503 cells towards neutrophil lineage, measured by specific lineage specific surface markers. Expression of a PU.1 mutant lacking the glutamine rich region of the transactivation domain did not promote differentiation to neutrophils. Moreover, the glutamine rich and acidic motifs are not necessary for the expression of the CD11b. Despite the independence of CD11b expression from both transactivation domain motifs, its expression was dependent on PU.1 DNA binding. Using cDNA microarray analysis, we identified PU.1 target genes that are regulated by specific domains of the PU.1 transcription factor. In our Affymetrix cDNA microarray analysis we identified over 1300 gene changes that are exclusively dependent on either the acidic domain or the glutamine rich region of the PU.1 transactivation domain for regulation. In addition, we found 33 PU.1- genes that are repressed by the PU.1 mutant lacking the acidic domain, but activated by the glutamine rich region PU.1 mutant, and 38 genes that are regulated in the opposite direction, thus allowing us to begin to identify the hierarchy of the PU.1-regulated gene cascade assocated with cellular function. Currently, we are investigating the biological significance of some of these differentially expressed target genes. For example, one of the identified genes, Neutrophil Elastase ( ELA2 ), is highly upregulated in the 503 cells expressing full length PU.1, or the PU.1 mutant lacking the acidic domain, but is profoundly downregulated in the 503 cell line expressing the glutamine rich region deletion mutant. It has been shown that ELA2 deficient mice are protected form acute promyelocytic leukemia. We are currently investigating the functional role of the PU.1-dependent regulation of ELA2 in our model system. In addition, we are identifying target genes that are regulated exclusively by other PU.1 domains, i.e. the PEST domain. Taken together, we identified and are analyzing the functional importance of specific target genes in PU.1-dependent myeloid differentiation that are regulated by different functional domains of the PU.1 transcription factor.