Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation

A steroid hormone responsive element (GRE/PRE), sufficient to confer glucocorticoid and progesterone inducibility when linked to a reporter gene, was used in band-shift assays to examine its molecular interactions with steroid hormone receptors. Both progesterone and glucocorticoid receptors bound directly and specifically to the GRE/PRE. The purine contact sites for both form A and form B chicken progesterone receptor, as well as those for rat glucocorticoid receptor, are identical. A peptide fragment produced in bacteria that primarily contain the DNA binding domain of the glucocorticoid receptor binds first to the TGTTCT half-site of the GRE/PRE, and a second molecule binds subsequently to the TGTACA (half-site) of the GRE/PRE in a cooperative manner. Utilizing the peptide fragment and the protein A-linked fragment, we demonstrated that the receptor interacts with its cognate enhancer as a dimer.     

Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements

We demonstrated previously that two molecules of steroid hormone receptor bound efficiently to a single hormone response element (GRE/PRE) of the tyrosine aminotransferase gene (Tsai et al., 1988). Here, we show that two tandemly linked GRE/PREs conferred progesterone inducibility synergistically to a heterologous TK-CAT fusion gene. Binding studies demonstrated that occupation of one GRE/PRE site by a progesterone receptor dimer increased the binding affinity of receptors for the second GRE/PRE site 100-fold. Thus, the observed synergistic induction of TK-CAT may result from cooperative binding of receptor dimers to the two GRE/PRE sites.     

Differential effects of nerve growth factor and dexamethasone on herpes simplex virus type 1 oriL- and oriS-dependent DNA replication in PC12 cells.

The herpes simplex virus type 1 (HSV-1) genome contains three origins of DNA replication, one copy of oriL and two copies of oriS. Although oriL and oriS are structurally different, they have extensive nucleotide sequence similarity and can substitute for each other to initiate viral DNA replication. A fundamental question that remains to be answered is why the HSV-1 genome contains two types of origin. We have recently identified a novel glucocorticoid response element (GRE) within oriL that is not present in oriS and have shown by gel mobility shift assays that purified glucocorticoid receptor (GR), as well as GR present in cellular extracts, can bind to the GRE in oriL. To determine whether glucocorticoids and the GRE affect the efficiency of oriL-dependent DNA replication, we performed transient DNA replication assays in the presence and absence of dexamethasone (DEX). Because HSV-1 is a neurotropic virus and establishes latency in cells of neural origin, these tests were conducted in PC12 cells, which assume the properties of sympathetic neurons when differentiated with nerve growth factor (NGF). In NGF-differentiated PC12 cells, oriL-dependent DNA replication was enhanced 5-fold by DEX, whereas in undifferentiated cells, DEX enhanced replication approximately 2-fold. Notably, the enhancement of oriL function by DEX was abolished when the GRE was mutated. NGF-induced differentiation alone had no effect. In contrast to oriL, oriS-dependent DNA replication was reduced approximately 5-fold in NGF-differentiated PC12 cells and an additional 4-fold in differentiated cells treated with DEX. In undifferentiated PC12 cells, DEX had only a minor inhibitory effect (approximately 2-fold) on oriS function. Although the cis-acting elements that mediate the NGF- and DEX-specific repression of oriS-dependent DNA replication are unknown, a functional GRE is critical for the DEX-induced enhancement of oriL function in NGF-differentiated PC12 cells. The enhancement of oriL-dependent DNA replication by DEX in differentiated PC12 cells suggests the possibility that glucocorticoids, agents long recognized to enhance reactivation of latent herpesvirus infections, act through the GRE in oriL to stimulate viral DNA replication and reactivation in terminally differentiated neurons in vivo.     

Functional serum response elements upstream of the growth factor-inducible gene zif268.

The zif268 gene, which encodes a protein with three typical zinc finger sequences, is induced in mouse 3T3 cells by serum, phorbol 12-myristate 13-acetate platelet-derived growth factor, and fibroblast growth factor. The induction is coordinate with that of c-fos. The 5'-flanking region of zif268 contains sequences that resemble known regulatory elements, including four CC(A or T)6GG sequences similar to the core serum response elements (SREs) found upstream of c-fos and actin genes. To determine whether the zif268 SRE-like elements mediate induction, CAT (chloramphenicol acetyltransferase) plasmids with different lengths of zif268 upstream sequences were tested for inducibility in 3T3 cells by serum, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. In addition, double-stranded oligonucleotides corresponding to each of the four zif268 putative SREs were tested individually for responsiveness when placed upstream of a thymidine kinase gene promoter. Each of the four SREs conferred inducibility by the agents tested, and multiple SREs resulted in greater inducibility than did a single element. Each of the zif268 SREs also competed with the c-fos SRE for binding by serum response factor present in HeLa cell nuclear extract. We conclude that the zif268 SRE-like sequences are functional and probably account for the coordinate induction of zif268 and c-fos.     

Functional characterization of an androgen response element in the first intron of the C3(1) gene of prostatic binding protein

We demonstrate that the 204 bp intronic gene fragment of C3(1), which has a specific in vitro affinity for the androgen receptor, is able to confer androgen responsiveness to a heterologous promoter. This characteristic is completely destroyed by a single G----T substitution, affecting a 5'-TGTTCT-3' element that closely resembles the consensus sequence of the glucocorticoid and progesterone response elements (GRE/PRE). In fact we could show that this androgen response element (ARE) also acts as a similarly weak GRE or PRE in T-47D cells.     

Multiple human papillomavirus type 16 glucocorticoid response elements functional for transformation, transient expression, and DNA-protein interactions.

We have previously shown that human papillomavirus type 16 (HPV-16) can efficiently transform primary baby rat kidney cells in the presence of the steroid hormones progesterone and the glucocorticoid dexamethasone. To study this effect of hormone, different combinations of the previously identified glucocorticoid response element (GRE) at nucleotide 7640 of HPV-16 and the other two GREs that we have recently identified, at nucleotides 7385 and 7474, were mutated. The previously described GRE and the other two GREs were shown to be functional for the induction of transformation by dexamethasone. In addition, transient assays in cervical HeLa cells demonstrated the functional importance of the three individual GREs. Assays for in vitro interaction demonstrated the specific binding of a 97-kDa protein, the glucocorticoid receptor, to both recently identified HPV-16 GREs.     

A silencer element in the first intron of the glutamine synthetase gene represses induction by glucocorticoids

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many tissues and cell lines, the synthetic glucocorticoid dexamethasone alone increases GS expression several fold. The direct response is mainly mediated by a cellular glucocorticoid receptor that, upon binding of the hormone, interacts with glucocorticoid responsive elements (GREs) of the gene. In cells of hepatocellular origin the response is mediated by a GRE located in the first intron of the gene. Surprisingly, hepatocytes do not respond to glucocorticoids with enhanced GS expression, despite the presence of an intact glucocorticoid receptor, which, in the same cells, stimulates expression of other genes such as tyrosine amino transferase. Reporter gene assays identified a sequence element downstream from the intronic GRE that inhibits the enhancement of expression by glucocorticoids. This silencer was designated GS silencer element of the rat. Gel mobility shift assays demonstrate the binding of a factor in hepatocyte nuclear extract. This yet unknown factor was designated GS silencer-binding protein. It is absent in FAO cells that respond to glucocorticoids with enhanced expression of GS and present in HepG2 cells that do not respond.