Purification of a protein doublet that binds to six TGG-containing sequences in the promoter for hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis, is transcribed at a relatively high level when cellular sterols are depleted and is repressed when sterols accumulate. We have previously reported that the regulatory region of the hamster reductase gene contains eight different sequences that bind nuclear proteins as determined by DNase I footprinting assays. We here report the purification of a single activity that accounts for six of these footprints. This activity was found in a doublet of proteins (designated reductase promoter factor 1, RPF-1) that have apparent molecular weights of 33,000 and 35,000. They were isolated by DNA affinity chromatography using oligonucleotides corresponding to either of two footprinted sequences. The 33- and 35-kDa species were present as monomers, as indicated by gel filtration and gradient ultracentrifugation. Oligonucleotides corresponding to any one of the six footprinted sequences prevented the binding of RPF-1 to all of the other sequences, indicating that all six bind to a single site in RPF-1. The only sequence shared by all six footprinted sequences is the trinucleotide, TGG, both of whose guanosines made contact with RPF-1, as determined by methylation interference assays. The footprinted sequence that binds RPF-1 with highest affinity contains the palindrome, TGG(N7)CCA, which conforms to the consensus sequence for binding NF-1, a nuclear protein that stimulates replication of adeno-virus-2. Purified RPF-1 was shown to bind to the adenovirus NF-1 binding site with high affinity. Although the apparent molecular weight of the RPF-1 doublet was lower than the molecular weight range for NF-1 proteins (52,000-66,000), it is likely that the 33-35-kDa doublet is derived from a larger NF-1-like protein as a result of proteolysis. We conclude that RPF-1 belongs to a group of TGG-binding proteins that includes NF-1 and other proteins previously described as CCAAT binding proteins. This protein binds to six sites in the promoter region for hamster 3-hydroxy-3-methylglutaryl CoA reductase, where its function remains to be determined.     

An evolutionarily conserved TNF-alpha-responsive enhancer in the far upstream region of human CCL2 locus influences its gene expression

Comparative cross-species genomic analysis has served as a powerful tool to discover novel noncoding regulatory regions that influence gene expression in several cytokine loci. In this study, we have identified several evolutionarily conserved regions (ECRs) that are shared between human, rhesus monkey, dog, and horse and that are upstream of the promoter regions that have been previously shown to play a role in regulating CCL2 gene expression. Of these, an ECR that was ~16.5 kb (-16.5 ECR) upstream of its coding sequence contained a highly conserved NF-κB site. The region encompassing the -16.5 ECR conferred TNF-α responsiveness to homologous and heterologous promoters. In vivo footprinting demonstrated that specific nucleotide residues in the -16.5 ECR were protected or became hypersensitive after TNF-α treatment. The footprinted regions were found to bind NF-κB subunits in vitro and in vivo. Mutation/deletion of the conserved NF-κB binding site in the -16.5 ECR led to loss of TNF-α responsiveness. After TNF-α stimulation, the -16.5 ECR showed increased sensitivity to nuclease digestion and loss of histone signatures that are characteristic of a repressive chromatin. Chromosome conformation capture assays indicated that -16.5 ECR physically interacts with the CCL2 proximal promoter after TNF-α stimulation. Taken together, these results suggest that the -16.5 ECR may play a critical role in the regulation of CCL2.     

Partial purification of a nuclear protein that binds to the CCAAT box of the mouse alpha 1-globin gene.

We enriched a fraction from nuclear extracts of murine erythroleukemia cells which contains a protein able to form stable complexes with the promoter region of the alpha 1-globin gene. Binding activity, which is present in mouse brain and a variety of cultured mouse and human cell lines, is not erythroid cell specific. Binding studies with alpha-globin gene promoter deletion mutants as well as DNase I footprinting and dimethyl sulfate protection studies showed that the factor bound specifically to the CCAAT box of the alpha 1 promoter. Enriched factor preparations exhibited weak binding to the promoter region of the beta maj-globin gene. This suggests that this protein could bind differentially to these two promoters in vivo. The enriched factor may be a ubiquitous nuclear protein involved in the differential regulation of the alpha 1- and beta maj-globin genes.     

The beta-globin dominant control region: hypersensitive site 2.

The Dominant Control Region (DCR) of the human beta-globin gene locus consists of four strong hypersensitive sites (HSS) upstream of the epsilon-globin gene. Addition of these sites confers copy number dependent expression on the human beta-globin gene in murine erythroleukaemia cells and transgenic mice, at levels comparable with the endogenous mouse globin genes. We have shown previously that a 1.9 kb fragment comprising HSS 2 accounts for 40-50% of the full effect of the DCR. In this paper we describe a deletional analysis of HSS 2. We show that a 225 bp fragment is sufficient to direct high levels of expression of the human beta-globin gene which is copy number dependent and integration site independent. This 225 bp fragment overlaps the major region that is hypersensitive 'in vivo'. DNase I footprinting shows the presence of four binding sites for the erythroid specific protein NF-E1; the three other footprinted regions display a remarkable redundancy of the sequence GGTGG and bind a number of proteins including Sp1 and the CACC box protein. The significance of these results for the regulation of globin gene expression is discussed.     

Delineation of AbrB-binding sites on the Bacillus subtilis spo0H, kinB, ftsAZ, and pbpE promoters and use of a derived homology to identify a previously unsuspected binding site in the bsuB1 methylase promote.

DNase I footprinting experiments showed that AbrB binds to the regulatory regions of the spo0H, kinB, ftsAZ, and pbpE genes. A conserved motif was found in these and other AbrB-binding sites. A search for Bacillus subtilis DNA sequences containing this motif led to the prediction that AbrB would bind to the promoter controlling the bsuB1 methylase gene. DNase I footprinting experiments confirmed this prediction.