Functional analysis of the trans-acting factor binding sites of the mouse alpha-fetoprotein proximal promoter by site-directed mutagenesis.

The trans-acting factors of the mouse alpha-fetoprotein proximal promoter (-202 base pairs) are aligned as follows: regions Ia (HNF-1), Ib (C/EBP), II (NF-1 or C/EBP), II' (NF-1 or HNF-1), III (NP-III), IV (NP-IV), Va (NP-Va), and Vb (C/EBP). Site-specific mutation abolished protein binding to the corresponding mutated site with the exception of the NF-1 site, in which mutation causes partial protection. Transient expression analyses indicate that chloramphenicol acetyl-transferase (CAT) activity is reduced by mutations in regions Ia, II', Ib, II, and IV. Mutation of region III causes an increased activity and mutation of regions Va and Vb shows a slight inhibitory effect. Linking alpha-fetoprotein enhancer I to the wild type promoter resulted in a 12-fold stimulation of CAT activity. The activity of promoters with mutated C/EBP-binding sites (Ib, II, and Vb), was slightly above controls, indicating that enhancer I can reverse the effect of these mutations. Inhibition or stimulation of promoter activity resulting from mutations of the HNF-1 or NP-III binding sites, respectively, persisted when enhancer I was linked to the promoters, indicating that enhancer I cannot rescue these mutations. Mutation of both HNF-1-binding sites resulted in greater than 90% inhibition of CAT expression with and without enhancer I, indicating these sites are essential for promoter activity. The stimulation of promoter activity by mutation of the NP-III site suggests that this site may be essential for repression or attenuation of the alpha-fetoprotein gene. Our studies indicate that regulation of the alpha-fetoprotein gene requires the combinatorial effect of multiple cis- and trans-acting elements in the proximal promoter and that enhancer I may provide a factor(s) that specifically rescue the promoter from the inhibitory effect of mutation in the C/EBP-binding sites.     

Structural and functional characterizations of the 5'-flanking region of the mouse glucagon receptor gene: comparison with the rat gene

A putative proximal promoter was defined previously for the mouse glucagon receptor (GR) gene. In the present study, a distal promoter was characterized upstream from a novel non-coding exon revealed by the 5'-rapid amplification of cDNA ends from mouse liver tissue. The 5'-flanking region of the mouse GR gene was cloned up to 6 kb and the structural organization was compared to the 5' untranslated region of the rat gene cloned up to 7 kb. The novel exon, separated by an intron of 3.8 kb from the first coding exon, displayed a high homology (80%) with the most distal of the two untranslated exons found in the 5' region of the rat GR gene. The mouse distal promoter region, extending up to -1 kb from the novel exon, displayed 85% identity with the rat promoter. Both contain a highly GC-rich sequence with five putative binding sites for Sp1, but no consensus TATA or CAAT elements. To evaluate basal promoter activities, 5'-flanking sequences of mouse or rat GR genes were fused to a luciferase reporter gene and transiently expressed in a mouse and in a rat cell line, respectively or in rat hepatocytes. Both mouse and rat distal promoter regions directed a high level of reporter gene activity. Deletion of the Sp1 binding sites region or mutation of the second proximal Sp1 sequence markedly reduced the distal promoter activity of the reporter gene. The mouse proximal promoter activity was 2- to 3-fold less than the distal promoter, for which no functional counterpart was observed in the similar region of the rat gene.     

Deletion analysis of the mouse alpha 1(III) collagen promoter.

A chimeric gene was constructed by fusing the DNA sequences containing the 5' flanking region of the mouse alpha 1(III) collagen gene to the coding sequence of the bacterial chloramphenicol acetyltransferase (CAT) gene. Transient transfection experiments indicated that the alpha 1(III) promoter is active in NIH 3T3 fibroblasts and BC3H1 smooth muscle cells. The activity of the alpha 1(III) collagen promoter-CAT plasmid is stimulated approximately ten fold by the presence of the SV40 enhancer element. Removing sequences upstream of -200 stimulates the activity of the chimeric gene eight fold. Further deletion analysis identified sequences located between -350 and -300 that were instrumental in repressing the activity of the promoter. This 50 bp region contains a direct repeat sequence that may be involved in the regulation of the mouse alpha 1(III) collagen gene. Truncating the alpha 1(III) promoter to -80 further stimulated expression. We propose that the positive regulatory elements of this gene appear to be located within the first 80 bp of the promoter, whereas elements located further upstream exert a negative effect on the expression of the gene. Regulation of the alpha 1(III) gene contrasts with that of the alpha 2(I) collagen gene, which appears to be regulated by several positive elements located in various regions of the promoter.     

Isolation and characterization of the QM promoter.

This report describes the isolation, sequencing and preliminary characterization of the first 1 kb of the 5'-regulatory region of the human QM gene. This region and the 5' -half of the transcribed region of the QM gene are enriched for C and G nucleotides with no bias against CpG dinucleotides--indicative of a CpG island. Several consensus GC boxes are present within the sequence. Most are clustered at the distal end, with one site present in the proximal 200 bp of the promoter. Electrophoretic mobility shift experiments and luciferase assays done in insect cells transfected with an Sp1 expression construct suggest that most of these sites can bind Sp1 or a closely related factor. In addition, the promoter is shown to be responsive to cAMP via a response element (CRE) in the proximal promoter. Studies with 5'-end and internal deletion mutants suggest that elements in the distal promoter exert their positive effect through interactions with a proximal element(s). Candidate proximal elements include the proximal GC box and a 43 bp region between a KpnI site (at -182) and a Smal site (at -139).     

Pancreatic beta cell-specific transcription of the pdx-1 gene. The role of conserved upstream control regions and their hepatic nuclear factor 3beta sites

To identify potential transactivators of pdx-1, we sequenced approximately 4.5 kilobases of the 5' promoter region of the human and chicken homologs, assuming that sequences conserved with the mouse gene would contain critical cis-regulatory elements. The sequences associated with hypersensitive site 1 (HSS1) represented the principal area of homology within which three conserved subdomains were apparent: area I (-2694 to -2561 base pairs (bp)), area II (-2139 to -1958 bp), and area III (-1879 to -1799 bp). The identities between the mouse and chicken/human genes are very high, ranging from 78 to 89%, although only areas I and III are present within this region in chicken. Pancreatic beta cell-selective expression was shown to be controlled by mouse and human area I or area II, but not area III, from an analysis of pdx-1-driven reporter activity in transfected beta- and non-beta cells. Mutational and functional analyses of conserved hepatic nuclear factor 3 (HNF3)-like sites located within area I and area II demonstrated that activation by these regions was mediated by HNF3beta. To determine if a similar regulatory relationship might exist within the context of the endogenous gene, pdx-1 expression was measured in embryonic stem cells in which one or both alleles of HNF3beta were inactivated. pdx-1 mRNA levels induced upon differentiation to embryoid bodies were down-regulated in homozygous null HNF3beta cells. Together, these results suggest that the conserved sequences represented by areas I and II define the binding sites for factors such as HNF3beta, which control islet beta cell-selective expression of the pdx-1 gene.     

Sequence of the human CALM II calmodulin gene promoter region

A clone containing a portion of the promoter region for the human bona fide CALM II gene was isolated using a human Promoter Finder DNA Walking Kit. This promoter region contains, putatively, a GC box (common in housekeeping genes), a CRE-binding site, a TATA like box and AGGGA sequences. The latter are reported to be present in genes for Ca2+ binding genes. This human promoter region exhibits overall 85% sequence identity to the corresponding region of the rat CALM II promoter but shows no identity to the corresponding region of the human CALM I or CALM III promoters.     

Structure-function analysis of the rat prolactin promoter: phasing requirements of proximal cell-specific elements

Expression of PRL, a member of the GH family of genes, is restricted to the lactotroph cells of the anterior pituitary. The proximal promoter of the rat PRL (rPRL) gene contains four factor-binding sites. Three nonadjacent elements, footprints (FP) I, III, and IV, are separated by an integral number of helical turns and bind a pituitary-specific factor, LSF-1. FP II binds another factor present in pituitary and nonpituitary cells. The mechanisms by which DNA-bound proteins influence RNA polymerase-II activity over large distances are not fully understood, but protein-protein interactions, with looping of intervening DNA, may bring distant sites into close proximity. Here, we demonstrate, using protein titration studies, that LSF-1 binds to the most proximal FP I element with the highest affinity, whereas it binds the more distal elements, FP III and FP IV, with progressively lower affinities. Time-course and salt-sensitivity studies reveal that binding of LSF-1 to all three pituitary-specific rPRL promoter sites occurs rapidly (less than or equal to 1 min) and requires fairly high salt concentrations (greater than or equal to 300 mM KCl) to destabilize protein-DNA interactions. Moreover, once bound, the pituitary nuclear factor(s) induces a conformational change in rPRL DNA structure with greatly delayed kinetics (greater than 15 min) and at a different salt concentration than are required for simply factor binding. Taken together, these data suggest a model in which LSF-1 initially binds fairly rapidly to multiple nonadjacent elements and then interacts with itself or other DNA-bound proteins much more slowly, possibly looping or bending the rPRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)