DNase I hypersensitivity analysis of the human CCAAT enhancer binding protein epsilon (C/EBPepsilon) gene.
Human C/EBPepsilon 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/EBPepsilon gene is controlled, we cloned a large genomic region surrounding the C/EBPepsilon 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/EBPepsilon gene were examined in C/EBPepsilon 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/EBPepsilon gene. They were found only in hematopoietic cells and were especially prominent in C/EBPepsilon 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/EBPepsilon 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/EBPepsilon. 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/EBPepsilon that may be involved in regulating transcription of this gene.
Kubota
T
T
Division of Hematology/Oncology, Cedars-Sinai Research Institute, UCLA School of Medicine, 8700 Beverly Blvd., Los Angeles, CA 90048, USA.
Hirama
T
T
Verbeek
W
W
Kawano
S
S
Chih
D Y
DY
Chumakov
A M
AM
Taguchi
H
H
Koeffler
H P
HP
D000077556:Alitretinoin; D000970:Antineoplastic Agents / Q000494:pharmacology; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D003850:Deoxyribonuclease I / Q000276:immunology* / Q000378:metabolism*; D004342:Drug Hypersensitivity / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D005809:Genes, Regulator; D006801:Humans; D014158:Transcription, Genetic; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D022762:CCAAT-Enhancer-Binding Proteins; D000077556:Alitretinoin; D014212:Tretinoin; D003850:Deoxyribonuclease I