Molecular Cloning, Sequence, and Expression Patterns of the Human Gene Encoding CCAAT/Enhancer Binding Protein α (C/EBPα)

doi:10.1006/bbrc.1995.2439

Biochemical and Biophysical Research Communications

Volume 215, Issue 1, 4 October 1995, Pages 106-113

https://doi.org/10.1006/bbrc.1995.2439 Get rights and content

Abstract

The human gene encoding the transcription factor C/EBPα was isolated from an umbilical cord genomic library screened by low stringency hybridization. Two overlapping clones were characterized by restriction enzyme analysis and included 13.2 kb of the C/EBPα locus. The entire gene and 471 bp of the promoter were sequenced. The human C/EBPα gene is 2783 bp long and encodes a 356 amino acid long protein, which is the same in length as for rat C/EBPα. Compared to rat C/EBPα, there are two insertions of two amino acids and one deletion of four. The amino acid similarity between the two proteins is over 92%. The human C/EBPα gene was found to be expressed at the highest levels in placenta. High expression was also found in liver, lung, skeletal muscle, pancreas, small intestine, colon and in peripheral blood leukocytes. However, the expression was undetectable or very low in brain, kidney, thymus, testis and ovary. These results show that the human C/EBPα gene is expressed in a tissue restricted manner.

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The gene coding for CEBPα is located on chromosome 19q13.1. It has no introns and encodes 2783 base pairs (Birkenmeier et al., 1989; Antonson and Xanthopoulos, 1995). The mRNA-encoding CEBPα contains three AUGs sharing a common open reading frame (ORF), and alternative translation initiation can give rise to two protein isoforms.
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It is noted that the GATA3 has closely relationship with FoxA1 in hormone-dependent cancers, such as breast and prostate cancers, however, whether other members of GATA with similar function as GATA3, and the potential function of GATA/FoxAs in other carcinomas, are still unknown. The human transcription factor C/EBPα has been initially isolated from umbilical cord genomic library [166], while the mouse gene encoding C/EBP has three alleles which are identified on the restriction fragment length polymorphisms [167]. C/EBPα expression was tissue-specific and expressed highest in placenta while a relative high expression in tissues such as liver, lung, skeletal muscle and pancreas [166].
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2012, Biochimie
The human liver fatty acid binding protein (hFABP1) participates in cellular long-chain fatty acid trafficking and regulation of lipid metabolism and changes in hFABP1 are associated with an increased risk for type 2 diabetes, cardiovascular disease (CVD), and metabolic syndromes. Gene regulation of hFABP1 is not fully understood. Therefore, in the present study, the full length hFABP1 promoter (nucleotides −2125 to +51) and a series of truncated promoter regions were cloned. A luciferase reporter assay revealed that nucleotides −255 to +50 in the promoter region contained full of maximum hFABP1 promoter activity compared with the full length promoter. Furthermore high activity was shown when the plasmid was transfected into liver-derived cells such as the human hepatoblastoma cell line HepG2 and the hepatoma cell line Huh7. TFSEARCH and TESS programs were used to predict potential transcription factor binding sites. Two putative binding sites for the liver-enriched transcription factors hepatocyte nuclear factor 3β (HNF3β) and CCAAT/enhancer binding protein α (C/EBPα) were identified in the −255 nt to −155 nt hFABP1 promoter region. Site-directed mutagenesis of these two sites reduced dramatically hFABP1 promoter activity. In addition, the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) revealed that these binding sites were recognized by HNF3β and C/EBPα respectively. Overexpression of HNF3β and C/EBPα enhanced the transcription of hFABP1 and consequently improved the protein level of hFABP1 in HepG2 cells, while knockdown of HNF3β and C/EBPα showed the inverse effects. Taken together, the hFABP1 gene is highly transcribed in liver-derived cells, and regulated predominantly by liver-enriched transcription factors HNF3β and C/EBPα.
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2009, Journal of Molecular Diagnostics
Citation Excerpt :
Therefore, fragment analysis alone is not a thorough way to screen for CEBPA mutation in AML patients. Additional problems with the published fragment length assay are the lack of full coverage of the coding region of CEBPA (a GC-rich region of the gene is omitted), and the possibility of missing insertion or deletion mutations if they lie at the sites of PCR primer binding.16 Given that mutations are spread through all areas of the CEBPA gene, some clinically relevant mutations may be present in regions of the gene that are not analyzed using this method.
The goal of the study was to compare the performance of a fluorescence-based multiplex PCR fragment analysis to a direct sequencing method for detecting CEBPA mutations in patients with acute myeloid leukemia. Thirty-three samples were selected from a larger study of 107 cases of acute myeloid leukemia by screening for CEBPA mutations by sequence analysis. Of ten identified mutations, six (insertions and deletions) were detected by both sequencing and fragment methods. The fragment analysis method did not detect the remaining four base substitutions because the method cannot detect changes that result in identically sized products. The multiplex PCR fragment length analysis method therefore failed to detect substitution mutations accounting for 40% of total CEBPA mutations in our patient set. Our results indicate that fragment length analysis should not be used in isolation, and that direct sequencing is required to evaluate CEBPA gene mutational status in acute myeloid leukemia. A combination of the two assays may offer some advantages, chiefly in permitting more sensitive detection by fragment length analysis of insertions and deletions.

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Regular ArticleMolecular Cloning, Sequence, and Expression Patterns of the Human Gene Encoding CCAAT/Enhancer Binding Protein α (C/EBPα)

Abstract

Regular Article
Molecular Cloning, Sequence, and Expression Patterns of the Human Gene Encoding CCAAT/Enhancer Binding Protein α (C/EBPα)