Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Alx4 binding to LEF-1 regulates N-CAM promoter activity.

During murine embryogenesis, expression of the paired-like homeodomain protein Alx4 is restricted to tissues whose development depends on the expression of lymphoid enhancer factor-1 (LEF-1). Given the defects seen in hair follicle development in both LEF-1 and Alx4 knockout and mutant animals and the overlapping expression patterns, we predicted that LEF-1 and Alx4 might form physical complexes. We demonstrate here the interaction between LEF-1 and Alx4. This interaction is mediated through a specific proline-rich domain in the N-terminal region of Alx4 and requires the DNA-binding domain (HMG-box) of LEF-1. We also demonstrate that LEF-1 and Alx4 can bind simultaneously to adjacent sites on the neural cell adhesion molecule (N-CAM) promoter and that this binding alters N-CAM promoter activity. Furthermore, when expressed in primary mammary stromal cells, Alx4 decreases the expression of endogenous N-CAM protein. These results reveal a potential mechanism that gives rise to mesenchymal-specific activities of LEF-1.     

Characterization of human cytoglobin gene promoter region

Cytoglobin (CYGB) is a member of the vertebrate globin family together with hemoglobin, myoglobin and neuroglobin. Although the physiological function of CYGB is still unclear, spectroscopic studies show that CYGB contains a hexacoordinated heme pocket similar to other pentacoordinated globin proteins. CYGB shares a common phylogenetic ancestry with vertebrate myoglobin from which it diverged by duplication before the appearance of jawed vertebrates. The objective of this study is to identify the regulatory and promoter region of the human cytoglobin gene. 5' unidirectional deletion constructs demonstrated that the proximal promoter elements of human CYGB gene are located between -1113 to -10 relative to the translation start site. Site-directed mutagenesis showed that mutation of a c-Ets-1 motif at -1008 and Sp1 motifs at -400, -230 and -210 remarkably decreased the promoter activity. Gel shift assays confirmed the binding of DNA-nuclear proteins to these motifs. All these results indicate that CYGB gene expression can be up-regulated by c-Ets-1 and Sp1 motifs.     

Characterization of the human interleukin-2 receptor beta-chain gene promoter: regulation of promoter activity by ets gene products.

The interleukin-2 receptor (IL-2R) beta chain (IL-2R beta) is an essential signaling component of high- and intermediate-affinity IL-2Rs. Our laboratory previously reported that a DNA fragment containing 857 bp of 5'-flanking sequence of the human IL-2R beta gene exhibited promoter activity. We have now further characterized the promoter and delineated cis-acting regulatory regions. The region downstream of -363 is critical for basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and contains at least three enhancer-like regions. Among them, the -56 to -34 enhancer was the most potent and had high-level activity in two T-cell lines but not in nonlymphoid HeLaS3 and MG63 cells. This enhancer contains a GGAA Ets binding site which bound two Ets family proteins, Ets-1 and GA-binding protein in vitro. Mutation of the Ets motif strongly diminished both promoter and enhancer activities. We conclude that this Ets binding site plays a key role in regulating basal and phorbol myristate acetate-inducible IL-2R beta promoter activity and may also contribute to tissue-specific expression of the IL-2R beta gene.     

Characterization of the promoter region of Tetrahymena genes.

The regions between adjacent histone H3 and H4 genes, as well as portions of the genes, from 22 species of Tetrahymena have been amplified using the polymerase chain reaction and sequenced. Both histone genes are transcribed divergently with initiation occurring within the intergenic region, thus 2 sets of 22 homologous Tetrahymena promoters can be compared. A sequence comparison of these regions reveals a single putative promoter element, with a consensus sequence TATCCAATTCARA, present in front of each gene. This sequence contains a 'CCAAT' box, which also occurs at 8 locations preceding other ciliate genes. No other putative promoter sequences are found in front of these sets of histone genes. Sequences searched for include 'TATA' boxes, 'GC' boxes and other sequences suggested as putative promoter elements for ciliate genes. The coding strand immediately preceding ciliate genes is very high in A content and the consensus sequence at the site of protein synthesis is AAAATGG.