Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.     

MSX2 expression in the apical ectoderm ridge is regulated by an MSX2 and Dlx5 binding site.

The apical ectodermal ridge (AER) is a specialized ectodermal region essential for limb outgrowth. Msx2 expression patterns in limb development strongly suggest an important role for Msx2 in the AER. Our previous studies identified a 348-bp fragment of the chicken Msx2 gene with AER enhancer activity. In this study, the functions of four potential homeodomain binding TAAT sites in this enhancer were studied using transgenic mice and in vitro protein-DNA interactions. Transgenic studies indicate that the four TAAT sites are not redundant and that only the B-TAAT site is critical for AER enhancer activity. The expression patterns of Msx2 and Dlx5 genes in the AER suggest that they might be involved in the regulation of Msx2. In support of this hypothesis, we found that Msx2 and Dlx5 can bind to the B-TAAT site as well as to a fragment containing the D- and E-TAAT sites in the Msx2 AER enhancer sequences. (c)2002 Elsevier Science (USA).     

The protein kinase C pathway acts through multiple transcription factors to repress gonadotropin-releasing hormone gene expression in hypothalamic GT1-7 neuronal cells

The GnRH gene uses two well-defined regions to target expression to a small population of hypothalamic GnRH neurons: a 173-bp proximal promoter and a 300-bp enhancer localized at approximately -1800 to -1500 bp from the start site. Interaction of multiple factors with the GnRH enhancer and promoter is required to confer neuron-specific expression in vivo and in cells in culture. In addition, the expression of the GnRH gene is regulated by numerous neurotransmitters and hormones. Several of these effectors act through membrane receptors to trigger the protein kinase C pathway, and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a modulator of this pathway, has been shown to suppress GnRH gene expression through the promoter. We find that TPA suppresses expression through the GnRH enhancer as well as the promoter. In the enhancer, an Oct-1 binding site, a Pbx/Prep binding site, Msx/Dlx binding sites, and a previously unidentified protein-binding element at -1793, all contribute to TPA suppression. TPA treatment leads to decreased binding of Oct-1 and Pbx1a/Prep to their sites. However, a complex formed by GT1-7 nuclear extracts on the -1793 site is not affected by TPA treatment. It is known that cooperative interaction among multiple factors is necessary for GnRH gene expression; thus, one mechanism by which TPA suppresses GnRH gene expression is to disengage some of these factors from their cis-regulatory elements.     

Identification of the enhancer binding protein MBF-1 of the sea urchin modulator alpha-H2A histone gene

The modulator of the sea urchin alpha-H2A histone gene promoter is the only enhancer identified in the alpha-histone gene cluster. Binding of a single factor, denoted MBF-1, has previously detected in nuclear extracts from morula and gastrula embryos. Here, we describe the cloning of MBF-1 by screening a cDNA expression library with a tandem array of modulator binding sites. MBF-1 presents no similarity with other DNA binding proteins and contains nine Krüppel like Zn fingers. In vitro translated proteins and a factor from nuclear extracts interact with the modulator with identical specificity. In addition, MBF-1 expressed in human cells transactivates a reporter gene driven by an array of modulator sites. The DNA binding domain consists of the Zn fingers plus an adjacent basic region, while sequences in the N-terminal region mediates the transactivation function. MBF-1 is expressed in the unfertilized egg and in early and late developmental stages thus confirming that it is not a stage specific enhancer binding factor and that silencing of the alpha-H2A gene after hatching is not due to the lack of the transactivator.     

Structure and function of the enhancer 3'' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.     

Specific interactions of histone H1 and a 45 kilodalton nuclear protein with a putative matrix attachment site in the distal promoter region of a cell cycle-regulated human histone gene

Protein-DNA interactions within the promoter of a cell cycle-regulated human H4 histone gene were examined by binding of 5'-end-labeled DNA segments to Western blots of nuclear protein fractions. Specific protein interactions were observed with DNA segments located between -500 bp and -1,070 bp upstream of the ATG initiation codon and included a histone H1 binding segment flanked on both sides by binding sites for a 45 kD nuclear protein. This region of the gene contains a DNase I-sensitive site in the center (-720 to -820 bp), and sequence analysis revealed the presence of scaffold attachment sequences in the two flanking segments. Topoisomerase II consensus sequences and in vitro topoisomerase II cleavage sites were also detected in the two flanking segments. Our results suggest that the 45 kd nuclear protein may preferentially interact with these two segments of the H4 histone gene to mediate association with the nuclear matrix. The presence of negative regulatory elements in this putative matrix attachment region provides a basis for the speculation that such nuclear proteins are associated with alterations in gene-matrix interaction that are functionally related to gene expression.     

Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.     

Binding of a pancreatic nuclear protein is correlated with amylase enhancer activity.

The mouse amylase gene Amy-2.2 is expressed at high levels specifically in the acinar cells of the pancreas. The region between -172 and -110 of this gene includes sequence elements common to pancreas-specific genes. Nuclear proteins with specific affinity for this region were partially purified from rat pancreas. The consensus element of another pancreas-specific gene, elastase 1, competes for protein binding to the amylase sequences. Binding was localized by DNase I protection to the sequence -156 to -122. Site-directed mutagenesis of this sequence resulted in concomitant loss of protein binding and enhancer activity. Photo-affinity labelling of pancreatic nuclear extracts identified one predominant binding protein with a molecular weight of approximately 75 kDa. The data indicate that binding of this nuclear protein is essential for the enhancer activity of this pancreas-specific element.