Mutational analysis of repression and activation of the tyrP gene in Escherichia coli.

In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters. We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters. In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine. Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein. Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation. In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site.     

Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli.

Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes. In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent. An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix. These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes. In the case of tyrP, moving the upstream box also affected activation. Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream. Practically no activation was seen at intermediate positions, such as +7 and -4. It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site.     

Regulation of aroL expression by TyrR protein and Trp repressor in Escherichia coli K-12.

The promoter-operator region of the aroL gene of Escherichia coli K-12 contains three TYR R boxes and one TrpR binding site. Mutational analysis showed that TYR R boxes 1 and 3 are essential for TyrR-mediated regulation of aroL expression, while a fully functional TYR R box 2 does not appear to be essential for regulation. Regulation mediated by the TrpR protein required the TYR R boxes and TrpR site to be functional and was observed in vivo only with a tyrR+ strain. Under conditions favoring the formation of TyrR hexamers, DNase I protection experiments revealed the presence of phased hypersensitive sites, indicative of DNA backbone strain. This suggests that TyrR-mediated repression involves DNA looping. Purified TrpR protein protected the putative TrpR binding site in the presence of tryptophan, and this protection was slightly enhanced in the presence of TyrR protein. This result along with the in vivo findings implies that TyrR and TrpR are able to interact in some way. Inserting 4 bp between TYR R box 1 and the TrpR binding site results in increased tyrosine repression and the abolition of the tryptophan effect. Identification of a potential integration host factor binding site and repression studies of a himA mutant support the notion that integration host factor binding normally exerts a negative effect on tyrosine-mediated repression.     

Construction of a tyrP-lac operon fusion strain and its use in the isolation and analysis of mutants derepressed for tyrP expression.

The gene tyrP, which codes for a component of the tyrosine-specific transport system, has been localized on the Escherichia coli K-12 chromosome at min 42. A tyrP-lac operon fusion was constructed and used to isolate mutants that have altered expression from the tyrP promoter. All putative tyrP operator mutations were transferred onto a plasmid vector by recombination in vivo. Restriction enzyme analysis of the resultant plasmids suggests that some of these mutants arose from either an insertion or a deletion of DNA occurring within the region of DNA that contains the tyrP promoter.     

Molecular analysis of the TyrR protein-mediated activation of mtr gene expression in Escherichia coli K-12.

Expression of the mtr gene, which encodes a tryptophan-specific transport system in Escherichia coli K-12, is activated by the TyrR protein. Two TyrR protein binding sites (TYR R boxes) are positioned upstream of the -35 promoter region. Mutational and DNase protection studies indicate that TyrR protein binds preferentially to the TYR R box closest to the promoter, and this is essential for activation of gene expression. In the presence of tyrosine and ATP, a second TyrR molecule is able to cooperatively bind to the second box and cause a further increase in the level of activation.     

Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli

In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes.     

Insights into the quality of DnaA boxes and their cooperativity

Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replication-inactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitro-defined 9mer DnaA box consensus sequence (TT(A)/(T)TNCACA). The quality and cooperativity of the DnaA boxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the promoter-proximal position was determined: TTATCCACA had the highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C >A >or=G >T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes.     

High levels of glucose induce apoptosis in cardiomyocyte via epigenetic regulation of the insulin-like growth factor receptor

Diabetic hyperglycemia result in cardiovascular complications, but the mechanisms by which high levels of glucose (HG) cause diabetic cardiomyopathy are not known. We investigate whether HG-induced repression of insulin-like growth factor 1 receptor (IGF-1R) mediated by epigenetic modifications is one potential mechanism. We found that HG resulted in decreased IGF-1 receptor (IGF-1R) mRNA levels, and IGF-1R protein when compared with H9C2 rat cardiomyocyte cells incubated in normal glucose. HG also induced apoptosis of H9C2 cells. The effects of HG on reduced expression of IGF-1R and increased apoptosis were blocked by silencing p53 with small interference RNA but not by non-targeting scrambled siRNA. Moreover, HG negatively regulated IGF-1R promoter activity as determined by ChIP analysis, which was dependent on p53 since siRNA-p53 attenuated the effects of HG on IGF-1R promoter activity. HG also increased the association of p53 with histone deacetylase 1 (HDAC1), and decreased the association of acetylated histone-4 with the IGF-1R promoter. Furthermore, HDAC inhibitor relieved the repression of IGF-1R following HG state. These results suggest that HG-induced repression of IGF-1R is mediated by the association of p53 with the IGF-1R promoter, and by the subsequent enhanced recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF-1R promoter-p53 complex. In conclusion, our data demonstrate that HG decreases expression of IGF-1R and decreases the association of acetylated histone-4 with the IGF-1R promoter. These studies may help delineate the complex pathways regulating diabetic cardiomyopathy, and have implications for the development of novel therapeutic strategies to prevent diabetic cardiomyopathy by epigenetic regulation of IGF-1R.