Metal-dependent binding of a nuclear factor to the rat metallothionein-I promoter.

Genomic footprinting studies in vivo and experiments using synthetic metal regulatory elements (MREs) in vitro suggest protein binding to the MREs of the mouse and rat metallothionein I (MT-I) genes. Using gel-retardation assays of promoter fragments, we observe a cadmium-dependent binding factor for the rat MT-I promoter in rat hepatoma cells. This factor is present in extracts from both uninduced and cadmium-induced cells, but requires the presence of cadmium to bind to the promoter. The formation of a cadmium-dependent complex is competed by an oligonucleotide containing two MREs. This competition is lost when when one of the MREs is mutated, indicating a requirement for at least two MREs for binding of this factor. The cadmium-dependent factor dissociates more rapidly from the MT-I promoter than does a factor that binds to a consensus Sp1 site present on the same DNA fragment. UV crosslinking analysis using nuclear extracts from cadmium induced cells, in the presence of an oligonucleotide probe containing both 5-bromodeoxyuridine and 32P-deoxycytidine, identifies a 39 kDalton protein associated with the metal inducible complex.     

Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene

Background

In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes.

Principal Finding

We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser³⁵⁹, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser³⁵⁹. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable.

Conclusions

We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.     

A nuclear factor requires Zn2+ to bind a regulatory MRE element of the mouse gene encoding metallothionein-1

The metal ion requirement of nuclear proteins for binding to the metal regulatory element d(MREd) of the mouse gene encoding metallothionein-1 was investigated using an in vitro exonuclease III footprinting assay. The specific DNA-binding activity of the factor was inactivated by the chelating agents, EDTA and 1,10-phenanthroline. Binding activity was restored by Zn2+, but not by Cd2+. These results show that Zn2+ ions are a required component for specific in vitro DNA binding of the MREd-binding protein.     

Interactions of the nuclear matrix-associated steroid receptor binding factor with its DNA binding element in the c-myc gene promoter

Steroid receptor binding factor (RBF) was originally isolated from avian oviduct nuclear matrix. When bound to avian genomic DNA, RBF generates saturable high-affinity binding sites for the avian progesterone receptor (PR). Recent studies have shown that RBF binds to a 54 bp element in the 5'-flanking region of the progesterone-regulated avian c-myc gene, and nuclear matrix-like attachment sites flank the RBF element [Lauber et al. (1997) J. Biol. Chem. 272, 24657-24665]. In this paper, electrophoretic mobility shift assays (EMSAs) and S1 nuclease treatment are used to demonstrate that the RBF-maltose binding protei (MBP) fusion protein binds to single-stranded DNA of its element. Only the N-terminal domain of RBF binds the RBF DNA element as demonstrated by southwestern blot analyses, and by competition EMSAs between RBF-MBP and the N-terminal domain. Mass spectrometric analysis of the C-terminal domain of RBF demonstrates its potential to form noncovalent protein-protein interactions via a potential leucine-isoleucine zipperlike structure, suggesting a homo- and/or possible heterodimer structure in solution. These data support that the nuclear matrix binding site (acceptor site) for PR in the c-myc gene promoter is composed of RBF dimers bound to a specific single-stranded DNA element. The dimers of RBF are generated by C-terminal leucine zipper and the DNA binding occurs at the N-terminal parallel beta-sheet DNA binding motif. This complex is flanked by nuclear matrix attachment sites.     

Gibberellin treatment stimulates nuclear factor binding to the gibberellin response complex in a barley alpha-amylase promoter.

The promoters of a majority of cereal alpha-amylase genes contain three highly conserved sequences (gibberellin response element, box I, and pyrimidine box). Recent studies have demonstrated the functional importance of four regions that either coincide with or are immediately proximal to these three conserved elements as well as an upstream Opaque-2 binding sequence. In this study, we describe the characterization of nuclear protein factors from barley aleurone layers whose binding activity toward gibberellin response complex sequences from the barley low-pl alpha-amylase gene (Amy32b) promoter is stimulated by gibberellin A3 (GA3) treatment. Barley proteins isolated from crude nuclear extracts prepared from aleurone layers incubated with or without GA3 were fractionated by anion exchange fast protein liquid chromatography and studied using band shift assays, sequence-specific competitions, and DNase I footprinting. A GA3-dependent binding activity eluting at 210 mM KCl was shown to bind specifically to the gibberellin response element and the closely associated box I. DNase I footprinting with the proteins in this fraction indicated interactions with sequences in the gibberellin response element and box I. A second DNA binding activity eluting at 310 mM KCl was present constitutively in extracts prepared from tissues incubated both in the absence and in the presence of hormone. Proteins in this fraction were able to bind to many DNA sequences and, in general, were largely nonspecific. DNase I footprinting with the proteins in this fraction indicated a large area of protection with a single unoccupied region located at the 3' end of box I. The possible function of such an activity in hormone regulation of the alpha-amylase genes is discussed.     

Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol

Gel shift assays were employed to distinguish between the contribution of 17 beta-estradiol (E2) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis gave a dissociation constant of 390 +/- 40 pM for the E2-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperatively was not observed. A dissociation constant of 450 +/- 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-fold lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.     

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region.

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.