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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.

SUBMITTER: Giannola DM

PROVIDER: S-EPMC2193192 | biostudies-literature | 2000 Nov

REPOSITORIES: biostudies-literature

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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.

Giannola D M DM Shlomchik W D WD Jegathesan M M Liebowitz D D Abrams C S CS Kadesch T T Dancis A A Emerson S G SG

The Journal of experimental medicine 20001101 10

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. ...[more]

PMID: 11085749

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Project description:The hRFC (human reduced folate carrier) is ubiquitously but differentially expressed in human tissues and its levels are regulated by up to seven non-coding regions (A1, A2, A, B, C, D and E) and at least four promoters. For the hRFC-B basal promoter, regulation involves binding of Sp (specificity protein) transcription factors to a critical GC-box. By transiently transfecting HT1080 cells with 5'- and 3'-deletion constructs spanning 1057 bp of upstream sequence, a transcriptionally important region was localized to 158 bp flanking the transcriptional start sites. By gel shift and chromatin immunoprecipitation assays, USF (upstream stimulatory factor), Sp1 and Ikaros-related proteins were bound to consensus elements (one E-box, two GC-box and three Ikaros) within this region. The functional importance of these elements was confirmed by transient tranfections of HT1080 cells with hRFC-B reporter constructs in which they were mutated, and by co-transfections of Drosophila Mel-2 cells with wild-type hRFC-B promoter and expression constructs for USF1, USF2a, Sp1 and Ikaros 2 and 8. Both USF1 and Sp1 proteins transactivated the hRFC-B promoter. Sp1 combined with USF1 resulted in a synergistic transactivation. Identical results were obtained with USF2a. Ikaros 2 was a repressor of hRFC-B promoter activity whose effects were partly reversed by the dominant-negative Ikaros 8. In HT1080 cells, transfection with Ikaros 2 decreased endogenous hRFC-B transcripts, whereas USF1 and Sp1 increased transcript levels. Ikaros 2 also decreased reporter gene activity and levels of acetylated chromatin associated with the endogenous promoter. Collectively, these results identify transcriptionally important regions in the hRFC-B promoter that include multiple GC-box, Ikaros and E-box elements. Our results also suggest that co-operative interactions between transcription factors Sp1 and USF are essential for high-level hRFC-B transactivation and imply that these effects are modulated by the family of Ikaros proteins and by histone acetylation.

Dataset Information

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.

Publications

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.

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