Analysis of the Xenopus laevis CCAAT-enhancer binding protein alpha gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Abstract

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPalpha genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPalpha genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPalpha gene (xC/EBPalpha). The -1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPalpha or xC/EBPss. Deletion analysis showed that the -321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPalpha promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPalpha promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPalpha expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Interferon-γ Stimulates the Expression of the Inducible cAMP Early Repressor in Macrophages through the Activation of Casein Kinase 2

“…The amplification was carried out using the high fidelity Pwo DNA polymerase to minimize PCR-generated mutations (40,41), and this was confirmed by sequencing. The amplification product (ϳ530 bp) was purified, digested with BamHI and EcoRI, and subcloned into the pcDNA3 vector that had been previously digested with the same enzymes.…”

“…The recombinant protein was labeled with [ 35 S]methionine (40 Ci) to aid detection. Nuclear and whole cell extracts were prepared essentially as described previously (12,15,16,40,42). Protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin A, 10 g/ml aprotinin, 10 g/ml I-S soybean trypsin inhibitor) and DTT (0.5 mM) were added to all buffers before use.…”

“…The gels were then dried under vacuum and exposed to x-ray film. For antibody supershift assays, samples of nuclear or whole cell extracts were incubated with the appropriate antiserum for 30 min on ice prior to the addition of the radiolabeled probe (12,15,16,30,32,40,42). For competition assays, a 250 -500-fold molar excess of the double-stranded oligonucleotides was added to the samples of proteins prior to the addition of the radiolabeled probe (12, 15, 16, 30 -32, 40, 42).…”

xDnmt1 regulates transcriptional silencing in pre-MBTXenopusembryos independently of its catalytic function

“…We tested whether xDnmt1 can repress the activation of a minimum promoter that has four copies of the xSp1-binding site driving luciferase expression (p4xSp1-Lucif) (Kockar et al, 2001). Relative induction by xSp1 is reduced by 55% in the presence of…”

Liver-Enriched Transcription Factors in Liver Function and Development. Part II: the C/EBPs and D Site-Binding Protein in Cell Cycle Control, Carcinogenesis, Circadian Gene Regulation, Liver Regeneration, Apoptosis, and Liver-Specific Gene Regulation

“…Thus, direct binding of the C/EBP-␣ protein to the promoter of the C/EBP-␣ gene is not required for autoregulation (Timchenko et al, 1995). Studies with the Xenopus laevis C/EBP-␣ promoter showed that, in contrast to the human promoter and in common with the murine gene, the xenopus C/EBP-␣ promoter was subject to direct autoregulation (Kockar et al, 2001).…”