Properties of ets-1 binding to chromatin and its effect on platelet factor 4 gene expression.

Ets-1 is the founding member of the Ets family of transcription factors, containing a DNA binding domain, a pointed domain, and a transactivation domain (reviewed in references 39 and 54). The DNA binding domain is conserved among the members of the family and is referred to as the ETS domain. As a transcription factor, Ets-1 binds to sequences on the DNA and participates in transcriptional regulation. The consensus sequences for Ets-1 binding contain a common core trinucleotide, GGA. However, the binding specificities for different Ets family members are not well understood (17) and the binding sequences for Ets proteins are often collectively referred to as Ets sites or Ets elements. Although Ets-1 was first discovered as part of a fused oncogene in an avian retrovirus, multiple roles have been discovered for this widely expressed transcription factor, including tumorigenesis, vertebrate embryogenesis, and hematopoiesis (39). In the hematopoietic system, the importance of Ets-1 in the transcriptional regulation of lineage-specific genes has been reported for lymphocytes, eosinophils, and erythrocytes (28, 30, 40). Megakaryocytes develop from hematopoietic pluripotent stem cells, giving rise to circulating platelets. After lineage commitment, megakaryocytes enter a unique program of differentiation that includes lineage-specific gene expression and an endomitotic cell cycle, resulting in a mature cell with a large volume and a polyploid nucleus (38, 59). Platelets are generated by fragmentation of mature megakaryocytes. As an indispensable part of the differentiation program, megakaryocytes express a group of genes that are unique to the lineage. Most of the lineage-specific gene products are found in platelets and are important for proper platelet function (46). Two examples are platelet factor 4 (PF4), encoding a chemokine, and glycoprotein IIb (GPIIb), a subunit of the fibrinogen receptor. These genes are expressed from the early stages of lineage development (10, 23, 36). The rat PF4 promoter has been cloned and shown to drive lineage-specific expression in a transgenic mouse model (36), indicating that the elements defining lineage specificity are within this DNA segment. Megakaryocyte-specific promoters have been shown to be positively controlled by Ets elements (5, 12, 22, 24, 29, 37), like genes from other hematopoietic lineages (3, 16, 30, 31, 49). In a search for potential transcription regulators for megakaryocyte differentiation, it was found that Ets-1 is upregulated during megakaryopoiesis but not during erythropoiesis (45). Furthermore, overexpression of Ets-1 upregulates the megakaryocyte-specific promoter of the PF4 gene in HEL cells (29). In the GPIIb promoter, both Ets-1 and Fli-1 (an Ets family member) are involved in gene expression (22, 52). This suggested that Ets family members control lineage-specific gene expression in megakaryocytes and that Ets-1 may control the expression of some of these genes. Ets-1 is not unique to megakaryocytes; it is expressed in most of the hematopoietic lineages. It is unclear how a relatively ubiquitous factor regulates lineage-specific function. This problem is a recurring theme in mammalian development. In eukaryotic cells, DNA is present in the form of chromatin, which limits the access of many transcription factors and provides an extra level of control for gene transcription. This important aspect has not been fully explored for Ets-1. Hence, while identification of transcription factors that are coactivators of megakaryocyte-specific genes is important, it is also essential to study the binding properties of Ets-1 to chromatin and its relevance to gene expression. In this paper we focus on this latter aspect. Ets-1 binding to chromatin might require lineage-specific cofactors and/or chromatin-remodeling enzymes. Ets-1, like many transcription factors, is known to interact with other transcription factors and chromatin-modifying enzymes, such as MafB, USF-1, PAX5, and CBP/p300 (4, 13, 16, 25, 40, 41, 57). It is also possible that the binding of Ets-1 depends on the intrinsic affinity of Ets-1 for binding sites in DNA. Under such a model, the concentration of Ets-1 might determine which binding sites are occupied and then which genes are expressed. A similar mechanism has been found for the Ets family member PU.1 (11). In the present study we show for the first time that Ets-1 binds to endogenous megakaryocyte-specific promoters in cultured cells. Using a cell-free system, we found that chromatin does not inhibit Ets-1 binding to the PF4 and GPIIb promoters. Ets-1 binding is independent of lineage-specific cofactors and of energy-dependent chromatin remodeling. Ectopic expression of Ets-1 in cultured cells is sufficient for binding to lineage-restricted endogenous genes, in the apparent absence of lineage-specific cofactors. Ets-1 binding is not sufficient to activate lineage-specific genes in cultured cells. Our study is the first to demonstrate that the binding of Ets-1 to lineage-specific promoters does not require lineage-specific factors, and that the affinity of Ets-1 to naked DNA and chromatin is of the same order of magnitude. Via these investigations, we also identified in the PF4 promoter a new Ets-1 binding site that is important for PF4 expression and a DNase I-hypersensitive site which is programmed by Ets-1 binding to chromatin and which enhances promoter activity.