The Journal of Experimental Medicine
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Published online 20 November 2000.
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© The Rockefeller University Press, 0022-1007/2000/11/1479/ $5.00
The Journal of Experimental Medicine, Volume 192, Number 10, November 20, 2000 1479-1490

Original Article

Hematopoietic Expression of Hoxb4 Is Regulated in Normal and Leukemic Stem Cells through Transcriptional Activation of the Hoxb4 Promoter by Upstream Stimulating Factor (Usf)-1 and Usf-2

Diane M. Giannolaa, Warren D. Shlomchikd, Mithila Jegathesana, David Liebowitza, Charles S. Abramsa, Tom Kadeschc, Andrew Dancisa, and Stephen G. Emersona,b

a Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
b Department of Pediatrics, University of Pennsylvania, Philadelphia, Pennsylvania 19104
c Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104
d Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510
The University of Pennsylvania, Maloney 510, 3600 Spruce St., Philadelphia, PA 19010.215-349-5866215-662-2359

The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34+ bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3–10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20–50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34+ cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.

Key Words: homeobox genes • self-renewal • stem cells • USF • transcription

Abbreviations used in this paper: DMSO, dimethyl sulfoxide; D/N, dominant negative; EGFP, enhanced green fluorescent protein; EMSA, electrophoretic mobility shift assay; HOX, homeobox; HLH, helix loop helix; HUCS, human umbilical cord serum; HXRE, HOX response element; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MITF, microphthalmia transcription factor; PI3K, phosphatidylinositol 3-kinase; RACE, rapid amplification of cDNA ends; SCF, stem cell factor; TPO, thrombopoietin; USF, upstream stimulatory factor.

© 2000 The Rockefeller University Press


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