DNase I hypersensitivity analysis of the human CCAAT enhancer binding protein ε (C/EBPε) gene

Abstract

Human C/EBPε is a recently cloned member of the C/EBP family of transcriptional factors. Previous studies demonstrated that the expression of this gene is tightly regulated in a tissue-specific manner; it is expressed almost exclusively in myeloid cells. To understand the mechanism by which the expression of C/EBPε gene is controlled, we cloned a large genomic region surrounding the C/EBPε gene and performed a DNase I hypersensitivity analysis of this locus. These sites probably represent areas of binding of proteins modulating gene transcription. Hypersensitive (HS) regions in 30 kb of DNA surrounding the C/EBPε gene were examined in C/EBPε high-expressing (NB4, HL-60), low-expressing (Jurkat), very-low-expressing (KG-1), and non-expressing (K562) hematopoietic cells as well as in non-hematopoietic-non-expressing cells (MCF-7, DU 145, PC-3). Three HS sites were detected near the first exon of C/EBPε gene. They were found only in hematopoietic cells and were especially prominent in C/EBPε expressing cells, suggesting that these sites play an important role in transcribing the gene. These hypersensitive bands did not change when the cells were cultured with retinoids. Gel-shift assays using 200 bp of nucleotide sequences that encompassed the hypersensitive sites and nuclear extracts from NB4 and Jurkat cells (C/EBPε expressing) as well as K562 and MCF-7 cells (non-expressing) showed different retarded bands on gel electrophoresis. A fourth HS site, located about 11 kb upstream of exon 1, was found only in cells highly expressing C/EBPε. Two sites, one about 4.5 kb upstream of exon 1 and another about 8.5 kb downstream of exon 2, were positive only in non-expressing cell lines, suggesting that repressors may bind in these areas. Taken together, we have found six specific DNase I hypersensitive sites in the region of C/EBPε that may be involved in regulating transcription of this 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.