The Transcription Factor Sp1 Regulates the Myeloid-specific Expression of the Human Hematopoietic Cell Kinase (HCK) Gene through Binding to Two Adjacent GC Boxes within the HCK Promoter-Proximal Region

Abstract

The human hemopoietic cell kinase (HCK) is a member of the src family of protein tyrosine kinases specifically expressed in myeloid cells and to a minor extent in B-lymphoid cells. HCK expression is up-regulated at the transcriptional level during myeloid differentiation of hematopoietic cells. To elucidate the molecular basis of the differential HCK gene expression, the genomic region containing the HCK promoter was isolated and functionally characterized. A DNA fragment containing 101 base pairs of the 5-flanking sequence showed strong promoter activity in the macrophage cell line RAW264 but was inactive in the non-monocytic cell lines HUT-78 and NIH-3T3. Site-directed mutagenesis of the proximal promoter region showed that two GC-rich sequence elements are essential for transcriptional activity in myeloid cells. Electrophoretic mobility shift analysis using nuclear extracts obtained from RAW264 cells and from the promonocytic cell line U-937 revealed the formation of at least three distinct protein-DNA complexes at each of these sites, one of which was found to contain the transcription factor Sp1. Expression of a reporter gene linked to the ؊101 HCK promoter region was up-regulated by Sp1, but not by other members of the Sp1 family of transcription factors, in Drosophila Schneider cells. A synergistic effect on HCK promoter activity was observed at high concentrations of Sp1. Our results show that Sp1 plays an essential role in the regulation of the differential gene expression of the HCK gene. The human hematopoietic cell kinase (HCK)1 belongs to a family of cytoplasmic protein tyrosine kinases for which c-src serves as an archetype (1-4). All members of the family share common structural features, including a catalytic domain in their COOH-terminal half, two regulatory domains known as SH2 and SH3 domains, an N-terminal domain unique to each of the family members and a myristylation site at the amino terminus (5-7).The HCK protein interacts physically with numerous receptors (8 -10) and its catalytic activity is stimulated upon engagement of myeloid-specific receptors (11)(12)(13)(14). Inactivation of the HCK gene by homologous recombination in ES cells results in a mild phenotype in HCK-deficient mice, characterized by impaired phagocytosis in macrophages (15).Like many of the other src-related kinases, HCK expression has been detected in cells of the granulocytic and monocytic lineage (1, 2) and at low levels in B lymphocytes (16) and NK cells (17). The expression of the HCK gene is regulated at the level of transcription (18) and increases during myeloid differentiation of normal and leukemic hematopoietic cells. In addition, the expression of HCK is induced in normal human macrophages and bone marrow macrophages after exposure of cells to bacterial lipopolysaccaride (19 -21) while interferon-␥ treatment of the cells itself does not alter HCK expression, but primes macrophages toward a stronger induction of HCK gene expression after LPS stimulation (20,21).Transfections of reporter gene constructs into ...

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