DNase I hypersensitivity analysis of the human CCAAT enhancer binding protein ε (C/EBPε) gene
Introduction
The human CCAAT/enhancer binding protein (C/EBP) family of transcription factors includes C/EBPα [1], C/EBPβ [2], [3], [4], [5], [6], [7], C/EBPγ [8], C/EBPδ [3], [9], [10], C/EBPε [11], [12], and C/EBPζ [13]. These proteins have highly conserved C-terminal basic amino acid-rich regions and leucine zipper domains that are essential for DNA binding and dimerization, but they differ in their N-terminal regions [14], [15]. The members of this family are believed to bind to the same recognition sites on DNA having a consensus sequence: 5′-ATTGCGCAAT-3′ [16]. Previous studies have suggested that the C/EBP proteins interact with selected transcriptional factors including c-Myb [17], cAMP response element-binding protein (CREB) [18], Fos-Jun family [19], and NF-κB [20].
The human C/EBPε gene was recently cloned and localized to chromosome 14q11.2 [11], [12]. Differential splicing and use of alternative promoters can generate four proteins of 32, 30, 27, and 14 kDa [12], [21]. The functional significance of these variants remains unclear. Previous studies demonstrated that the expression of the C/EBP family was tightly regulated in a tissue-specific manner [22]. C/EBPα, C/EBPβ, C/EBPδ are all expressed during hematopoiesis, suggesting that these proteins may regulate a variety of hematopoietic related genes [23]. C/EBPε is exclusively expressed in the myeloid and T-lymphoid cell lineages, especially during granulocytic differentiation [11], [12], [21], [24]. A previous study using C/EBPε-deficient mice demonstrated that these mice developed normally but failed to generate functional neutrophils and eosinophils. These mice died of opportunistic infections, suggesting that C/EBPε may play a central role in myeloid differentiation [25]. In addition, studies have shown that retinoic acid (RA) can dramatically enhance the expression of C/EBPε in promyelocytic cell lines [21], [26], [27].
To understand the mechanism by which the expression of the C/EBPε gene is controlled, we have studied DNase I hypersensitivity of the C/EBPε locus. These hypersensitive areas probably represent sites of binding of proteins involved in modulating expression of the gene [28], [29]. Thus, by comparing DNase I hypersensitive sites (HS) in the vicinity of a gene, we will be able to delineate important protein-binding regions.
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Cell lines and culture conditions
Myeloid cell lines (HL-60, KG-1, NB-4), chronic myelogenous leukemia cell line (K562), lymphoid cell line (Jurkat) and non-hematopoietic cell lines (MCF-7, DU 145, PC-3) were used in this study. NB4 cells (promyelocytic leukemia cell line) were generously provided by Dr. M. Lanotte (St. Louis Hospital, Paris, France) [30]. KG-1 (early myeloblastic leukemia cell line) was established in our laboratory [31]. Other cell lines were obtained from the American Tissue Culture Collection (Rockville,
Genomic cloning of the C/EBPε locus
A human genomic lambda phage library was screened using the cDNA of C/EBPε as a probe. Twenty clones containing regions of the gene were obtained. Clones of 19.5 and 17.8 kb contained the upstream and downstream region of the gene, respectively. Together, these two genomic fragments totalling 30.5 kb encompassed the gene with its upstream and downstream regions. A restriction enzymatic map of the gene was made (Fig. 1). These results paralleled and expanded two previous studies [11], [12].
DNase I hypersensitivity analyses
We
Discussion
The expression of the C/EBPε gene is regulated in a strictly lineage-specific manner. It is almost exclusively expressed in myeloid cells as they undergo terminal differentiation. To understand the mechanism of cell type-specific expression, studies on cis-acting elements are important. To begin to dissect the regulation of expression of the C/EBPε gene, we examined the chromatin structure of this gene in cells that either do or do not express the gene. The chromatin of an actively transcribed
Acknowledgements
We thank A. Fritz Gombert and Hiroshi Kawabata (Cedars-Sinai Medical Center/UCLA School of Medicine) for their generous advice. We are grateful to Kim Burgin for her excellent secretarial help. This work was supported in part by National Institute of Health and US Department of Defense grants as well as the Parker Hughes, C. & H. Koeffler Fund, Horn Foundation and the Lymphoma Foundation of America. T. Kubota provided the concept, design, analysis and interpretation of the data and drafted the
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