Development of a Model for the Study of PU.1 Function in Primary Human Hematopoietic Cells.

PU.1 is one of the best-studied transcription factors governing hematopoiesis and has been shown to regulate positively differentiation of B-lymphocytes and granulocytes. PU.1 is also expressed in early erythroid progenitors and its interaction with GATA-1 was described to directly inhibit erythroid differentiation, since GATA-1 is the key regulator of erythropoiesis. In addition, the binding of GATA-1 to PU.1 was found to repress PU.1 dependent myeloid gene expression. In order to study more in detail the effect of PU.1 in primary human hematopoietic cell differentiation, we designed lentiviral vectors which allow PU.1 overexpression and PU.1 inhibition.

For PU.1 overexpression, we cloned the PU.1 cDNA into the pWPIR-ires-GFP bicistronic plasmid and verified by transient transfection in 293T cells the production of PU.1 mRNA and of right sized protein. We analyzed PU.1 function by co-transfection assays into 293T cells using the pWPIR-PU.1 vector and CAT reporter genes governed by PU.1 responsive elements. By CAT ELISA assay we observed a dramatic increase in OD. The production of PU.1 mRNA and protein in Hela cells was verified by stable transduction with complete lentivectors.

For PU.1 inhibition, we constructed a lentiviral vector encoding a siRNA specific for PU.1. After transduction of the K562 erythroleukemic cell line, both PU.1 mRNA and protein became undetectable, as verified by RT-PCR and Western blot, respectively, whereas GATA-1 mRNA and protein expression remained unchanged.

We tested both viral constructs in an in vitro culture system, in which CD34+ hematopoietic precursors obtained from bone marrow aspirates, differentiate into mature red cells under the influence of SCF, IL-3 and Epo or into mature granulocytes by stimulation with thrombopoietin, SCF and Flt-3L. Results for cultures with PU.1 transduced cells showed inhibition of erythroid cell differentiation by 40% ± 10% (mean of three experiments) and increased myeloid proliferation, whereas cultures with siPU.1 transduced cells showed no influence on erythroid cells and strong decrease of myeloid cell proliferation (50 – 60x) and differentiation (90 % decrease of CD13+ cells).

In conclusion, our model gives us the opportunity to test the function of PU.1 overexpression and/or inhibition in primary hematopoietic cells, to test the effect on target genes in various stages of differentiating precursors and the interaction with other transcription factors like GATA-1, and to analyze pathologic conditions like some forms of acute myeloid leukemia, where PU.1 was described to be mutated or downregulated.