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

Abstract

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.

Cha, a Basic Helix-Loop-Helix Transcription Factor Involved in the Regulation of Upstream Stimulatory Factor Activity

“…In agreement with this, USF-1 or USF-2 knockout mice have a diminished glucose response (21) and fatty acid synthase expression (22). Other genes such as follicle-stimulating hormone receptor (23), metallothionein (24), HOXB4 transcription factor (25), and the class II major histocompatibility complex (26) are regulated by USF-1. Additionally, USF overexpression has been found to inhibit growth in a number of cancer cell lines (27,28).…”

NF-Ya activates multiple hematopoietic stem cell (HSC) regulatory genes and promotes HSC self-renewal

“…TPO simulation of HSCs activates MAPK, which we and others have shown leads to induction of HOXB4 expression in both normal and K562 cells (6,27). Although a pivotal role of NF-Ya in TPO-induced HOXB4 activation is not yet known, MEK activation has itself been shown to up-regulate NF-Ya activity functioning on the promoter of other NF-Ya target genes such as GADD45 (28).…”

“…Recently, we demonstrated that the trimeric transcription factor NF-Y activates the HOXB4 promoter in cooperation with upstream͞ubiquitous stimulating factor 1 and 2 (USF1͞2) in normal and malignant hematopoietic cells (6,7). Studies from other laboratories suggested that NF-Y is also a potent inducer for numerous other genes whose function likely influences stem cell (SC) function, including HOXB7, JunB, p27, CDK1, MDR1, and TGF␤R-II (8)(9)(10)(11)(12)(13).…”

Hematopoietic cytokines, transcription factors and lineage commitment

“…Recently, the two essential DNA binding sites were identi®ed in the promoter region of HoxB4 gene functioning in both normal and malignant hematopoietic primitive cells, just upstream of its transcriptional starting site. One of them was a classical E box and served as the functional binding site for USF1 and USF2 (Giannola et al, 2000).…”