YY1 is a positive regulator of transcription of the Col1a1 gene.

Both cell-specific and ubiquitous transcription factors in fibroblasts have been identified as critical for expression of the Col1a1 gene, which encodes the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY1) binds to the Col1a1 promoter immediately upstream of the TATA box, and we examine the functional implications of YY1 binding for regulation of Col1a1 gene expression in BALBc/3T3 fibroblasts. The Col1a1 promoter region spanning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corresponding binding activity in nuclear extracts was supershifted using a YY1-specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex formation. Mutation analysis revealed two YY1 core binding sites at -40/-37 bp (YY1A) and, on the reverse strand, at -32/-29 bp (YY1B) immediately adjacent to the TATA box. In transfections using Col1a1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -222/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCMV-YY1 increased p350wt and p130wt activities by as much as 10-fold, whereas antisense YY1 decreased constitutive expression and blocked the increased activity due to pCMV-YY1 overexpression. The mTgTA constructs were devoid of activity, arguing for a requirement for cognate binding of the TATA box-binding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY1 could bind to the -56/-9 bp fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Col1a1 proximal promoter and functions as a positive regulator of constitutive activity in fibroblasts. Although YY1 is not sufficient for transcriptional initiation, it is a required component of the transcription machinery in this promoter.

Cytomegalovirus IE2 protein stimulates interleukin 1beta gene transcription via tethering to Spi-1/PU.1.

Potent induction of the gene coding for human prointerleukin 1beta (il1b) normally requires a far-upstream inducible enhancer in addition to a minimal promoter located between positions -131 and +12. The transcription factor Spi-1 (also called PU.1) is necessary for expression and binds to the minimal promoter, thus providing an essential transcription activation domain (TAD). In contrast, infection by human cytomegalovirus (HCMV) can strongly activate il1b via the expression of immediate early (IE) viral proteins and eliminates the requirement for the upstream enhancer. Spi-1 has been circumstantially implicated as a host factor in this process. We report here the molecular basis for the direct involvement of Spi-1 in HCMV activation of il1b. Transfection of Spi-1-deficient HeLa cells demonstrated both the requirement of Spi-1 for IE activity and the need for a shorter promoter (-59 to +12) than that required in the absence of IE proteins. Furthermore, in contrast to normal, enhancer-dependent il1b expression, which absolutely requires both the Spi-1 winged helix-turn-helix (wHTH) DNA-binding domain and the majority of the Spi-1 TAD, il1b expression in the presence of IE proteins does not require the Spi-1 TAD, which plays a synergistic role. In addition, we demonstrate that a single IE protein, IE2, is critical for the induction of il1b. Protein-protein interaction experiments revealed that the wing motif within the Spi-1 wHTH domain directly recruits IE2. In turn, IE2 physically associates with the Spi-1 wing and requires the integrity of at least one region of IE2. Functional analysis demonstrates that both this region and a carboxy-terminal acidic TAD are required for IE2 function. Therefore, we propose a protein-tethered transactivation mechanism in which the il1b promoter-bound Spi-1 wHTH tethers IE2, which provides a TAD, resulting in the transactivation of il1b.

Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis.

Mammalian ribonucleotide reductase, which occupies a key position in the synthesis of DNA, is a highly controlled enzyme activity, because it is solely responsible for the de novo reduction of ribonucleoside diphosphates to their corresponding deoxyribonucleoside diphosphate forms, required for DNA synthesis. Ribonucleotide reductase consists of two dissimilar protein components often called M1 and M2, which are independently regulated during cell proliferation. The M1 component contains multiple effector binding sites and is responsible for the complex allosteric regulation of the enzyme, whereas the M2 protein contains nonheme iron and a unique tyrosyl-free radical required for ribonucleotide reduction. Since the reaction is rate limiting for DNA synthesis, ribonucleotide reductase plays an important role in regulating cell division, and hence, cell proliferation. There are many inhibitors of ribonucleotide reductase and perhaps the most valuable one from a cell biology, biochemistry, and clinical point of view is the hydroxamic acid, hydroxyurea. This drug has also been very useful as a selective agent for isolating a variety of mammalian mutant cell lines altered in ribonucleotide reductase gene expression. Regulatory, structural, and biological characteristics of ribonucleotide reductase are reviewed, including evidence that ribonucleotide reductase, particularly the M2 protein, has an important early role to play in tumor promotion. In addition, modifications in the expressions of genes altered in hydroxyurea-resistant mutants and cultured in the absence or presence of hydroxyurea are discussed, with emphasis on changes in M2 protein, M1 protein, and the iron-storage protein ferritin.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased ferritin gene expression is associated with increased ribonucleotide reductase gene expression and the establishment of hydroxyurea resistance in mammalian cells.

In the present study, we show that hydroxyurea-inactivated ribonucleotide reductase protein M2 has a destabilized iron center, which readily releases iron. In addition, evidence is presented which indicates that single or multistep selection for hydroxyurea resistance, in a variety of mammalian cell lines, leads to alterations in the expression of the gene for the iron storage protein, ferritin. In all hydroxyurea-resistant cell lines examined, including human, hamster, rat, and mouse, there was an elevation in ferritin heavy (H)- and/or light (L)-mRNA levels, but no change in the corresponding gene copy number. A detailed analysis of ferritin expression in a hydroxyurea-resistant mouse L cell line showed that when compared to its wild type counterpart, there was an increase in H subunit concentration but no significant change in L subunit levels. The increased H/L subunit ratio was not brought about by specific changes in the rates of ferritin subunit biosynthesis, but rather resulted from changes in the post-translational stability of H subunits relative to L subunits in the resistant cell line compared to its parental wild type. Also, we show that treatment of cells with hydroxyurea results in an increased rate of ferritin biosynthesis in the absence of changes in H- or L-mRNA levels. These results indicate that the development of even low level hydroxyurea resistance in mammalian cells may require alterations in ferritin gene expression, and they show an interesting relationship between the expressions of two highly regulated activities, ribonucleotide reductase and ferritin.

Transient elevation of ribonucleotide reductase activity, M2 mRNA and M2 protein in BALB/c 3T3 fibroblasts in the presence of 12-O-tetradecanoylphorbol-13-acetate.

A rapid elevation of ribonucleotide reductase activity was observed with BALB/c 3T3 fibroblasts within 1/2 to 1 hour treatment with 0.1 microM 12-O-tetradecanoylphorbol-13-acetate (TPA). This increase in activity was transient, and returned to about normal levels within 24 to 48 hours. Northern analysis of the two components of ribonucleotide reductase showed a slight transient elevation of M1 mRNA and a marked transient elevation of M2 mRNA after 1/2 hour TPA treatment. As a positive control, ornithine decarboxylase message levels were also observed to be transiently elevated following identical treatment with TPA. Western blot analysis with M1 and M2 specific monoclonal antibodies indicated that the increase in ribonucleotide reductase activity was primarily due to the transient elevation of the M2 but not the M1 protein during treatment with 0.1 microM TPA. This first demonstration that the tumor promotor, TPA, can cause rapid and transient alterations in ribonucleotide reductase suggests that the enzyme, particularly the M2 component, may play an important role in the critical events involved in the process of tumor promotion.

Molecular mechanisms responsible for the drug-induced posttranscriptional modulation of ribonucleotide reductase levels in a hydroxyurea-resistant mouse L cell line.

Ribonucleotide reductase, which catalyzes the formation of deoxyribonucleotides from ribonucleoside diphosphate precursors, is the rate-limiting enzyme in DNA synthesis. The enzyme consists of two nonidentical subunits called M1 and M2, both of which are required for activity. Hydroxyurea, a specific inhibitor of DNA synthesis, acts by destroying the unique tyrosyl free radical of protein M2. Previously, we have described a mouse L cell line which exhibited a stable resistance to high concentrations of hydroxyurea. This mutant cell line contains elevated quantities of both proteins M1 and M2 as a result of corresponding increases in the levels of mRNAs for both subunits. Interestingly, both M1 and M2 protein levels were further elevated when mutant cells were cultured in the presence of hydroxyurea, and this elevation was not accompanied by increases in their corresponding mRNAs. These results indicated that hydroxyurea can modulate ribonucleotide reductase expression posttranscriptionally. In this report, we show that the level of both subunits of ribonucleotide reductase responds to hydroxyurea in a drug concentration dependent manner. Furthermore, results from kinetic studies indicate that protein M2 levels rise much more rapidly than protein M1. Pulse-chase experiments indicated that the half-lives of both the M1 and M2 polypeptides are increased by approximately 2-fold when the mutant cells are cultured in the presence of hydroxyurea. We also present evidence indicating that exposure of these cells to hydroxyurea leads to a relatively slow but specific increase in the rate of biosynthesis of both proteins M1 and M2, as assayed by pulse labeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanisms of drug resistance involving ribonucleotide reductase: hydroxyurea resistance in a series of clonally related mouse cell lines selected in the presence of increasing drug concentrations.

Mammalian ribonucleotide reductase is a highly regulated, rate-limiting activity responsible for converting ribonucleoside diphosphates to the deoxyribonucleotide precursors of DNA. The enzyme consists of two nonidentical proteins often called M1 and M2, both of which are required for activity. Hydroxyurea is an antitumor agent which inhibits ribonucleotide reductase by interacting with the M2 component specifically at a unique tyrosyl free radical. To obtain further information about drug resistance mechanisms, we have used M1 and M2 complementary DNAs and monoclonal antibodies to investigate the properties of a series of clonally related drug-resistant mouse cell lines, selected by a step-wise procedure for increasing levels of resistance to the cytotoxic effects of hydroxyurea. Several interesting mechanisms have been identified. Each successive drug selection step leading to the isolation of highly resistant cells was accompanied by stable elevations in cellular resistance and ribonucleotide reductase activities. The changes that occurred at each step involved the M2 component. A very early event, occurring at the first step in the selection process, was the amplification of the M2 gene accompanied by an increase in M2 messenger RNA. Although cellular resistance and M2 protein levels increased significantly during drug selection, only a modest change in M2 gene copy number was observed after the initial selection step. Analysis of wild type, moderately resistant, and highly resistant cells indicated that, in addition to M2 gene amplification, posttranscriptional modification also occurred during drug selection. This second mechanism was not due to alterations in protein M2 half-life, but involved an increase in translational efficiency. By increasing the rate of M2 synthesis, without altering degradation rates, resistant cells were able to accumulate high levels of this key regulatory protein. Cells selected for the ability to proliferate in concentrations of drug as high as 4 mM exhibited changes that involved M2, without detectable changes to M1. These results provide further evidence that M1 and M2 levels are controlled by different mechanisms in mammalian cells. Eventually, however, cells required an elevation in the M1 protein, as well as the M2 protein, to survive in a hydroxyurea concentration of 5 mM. These results illustrate the complexity of the drug-resistant phenotype and provide further information about the molecular processes that lead to the development of cells resistant to low, intermediate, and high concentrations of hydroxyurea.

Reversion of hydroxyurea resistance, decline in ribonucleotide reductase activity, and loss of M2 gene amplification.

A key rate-limiting reaction in the synthesis of DNA is catalyzed by ribonucleotide reductase, the enzyme which reduces ribonucleotides to provide the deoxyribonucleotide precursors of DNA. The antitumor agent, hydroxyurea, is a specific inhibitor of this enzyme and has been used in the selection of drug resistant mammalian cell lines altered in ribonucleotide reductase activity. An unstable hydroxyurea resistant population of mammalian cells with elevated ribonucleotide reductase activity has been used to isolate three stable subclones with varying sensitivities to hydroxyurea cytotoxicity and levels of ribonucleotide reductase activities. These subclones have been analyzed at the molecular level with cDNA probes encoding the two nonidentical subunits of ribonucleotide reductase (M1 and M2). Although no significant differences in M1 mRNA levels or gene copy numbers were detected between the three cell lines, a strong correlation between cellular resistance, enzyme activity, M2 mRNA and M2 gene copies was observed. This is the first demonstration that reversion of hydroxyurea resistance is directly linked to a decrease in M2 mRNA levels and M2 gene copy number, and strongly supports the concept that M2 gene amplification is an important mechanism for achieving resistance to this antitumor agent through elevations in ribonucleotide reductase.