Interferon-gamma-inducible regulation of the human invariant chain gene

Interferon-gamma (IFN-gamma) regulates a variety of immunoregulatory functions through the induction of a specific set of IFN-gamma response genes. This includes the invariant chain associated with the major histocompatibility complex class II molecules. To investigate the mechanism involved in the invariant chain (In) response to IFN-gamma we constructed chloramphenicol acetyltransferase (CAT) hybrid genes in which the CAT gene is under the control of the In promoter. The glioblastoma cell line, U-373 MG, transfected with a CAT construct having the In promoter sequence -790 to +1 bp showed over 3-fold increased CAT activity when treated with IFN-gamma indicating that this region confers IFN-gamma responsiveness to the CAT gene. The IFN-gamma response element in the promoter was further sublocalized to the region -120 to -61 base pairs (bp). This region contains homology to the interferon-stimulated response elements identified in other IFN responsive genes. By gel shift analyses, an IFN-gamma-induced sequence-specific DNA-binding factor was identified. This induced complex binds to an oligonucleotide corresponding to -107 to -79 bp of the In promoter. Mutations of this binding site at -94 and -92 bp drastically decreased binding of the constitutive and IFN-gamma-induced complexes. This IFN-gamma induced factor also binds to an oligonucleotide corresponding to -91 to -62 bp of the interferon-beta (IFN-beta) gene promoter, a region necessary for the induction of the IFN-beta gene by virus and double-stranded RNA. This binding specificity is characteristic of a family of DNA binding factors that bind both the interferon-stimulated response elements and the IFN-beta gene promoter.     

Identification of estrogen-responsive DNA sequences by transient expression experiments in a human breast cancer cell line.

The expression of a hybrid gene formed by the promoter region of the Xenopus laevis vitellogenin gene B1 and the CAT coding region is regulated by estrogen when the gene is transfected into hormone-responsive MCF-7 cells. Furthermore, the 5' flanking region of the gene B1 alone can confer inducibility to heterologous promoters, although to a varying extent depending on the promoter used. Deletion mapping of he vitellogenin hormone-responsive sequences revealed that a 13 bp element 5'-AGTCACTGTGACC-3' at position -334 is essential for estrogen inducibility. We have shown previously that this 13 bp element is present upstream of several liver-specific estrogen-inducible genes.     

Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.     

Upstream regulatory elements of murine alpha 4-interferon gene confer inducibility and cell type-restricted expression

We have identified and functionally characterized DNA sequences that are required for the inducible and cell-restricted expression of the murine alpha 4-interferon gene. Hybrid plasmids in which the alpha 4 promoter region or its 5' deletions were inserted upstream of the CAT gene were constructed, and the expression of these hybrid genes was studied in mouse L-cells both in permanent and transient assays with comparable results. Inducible expression was not affected by deletions up to -109; however, when the deletion was extended to -96, inducibility by Newcastle disease virus was abolished; however, this hybrid plasmid was expressed constitutively. Further deletion to -88 did not permit either constitutive or inducible expression. Insertion of the 35-base pair-long sequence (-109 to -75 base pairs) from the alpha 4 promoter region 5' of the minimal alpha 4 or human immunodeficiency virus promoter region, conferred inducibility to these two inactive promoters. The 5' deleted hybrids or plasmids containing the inducible element were induced only at low levels in transfected NIH/3T3 cells that do not express endogenous alpha 4 gene efficiently, indicating that the inducible region also determines the cell-specific expression. A tandem repeat of AGTGAA, which is present in the -109 to -88 region of alpha 4 in two copies, showed both basal levels of expression and inducibility in L-cells, while its analogue AATGAA was highly inducible but was not expressed constitutively. The inducibility of the synthetic hexamer repeats did not show cell type-restricted expression, suggesting that their response does not fully reflect the range of expression observed for the inducible region and the endogenous alpha genes.     

A potato Sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects.

To examine which sequences are involved in regulating the potato sucrose synthase gene Sus3-65, we examined a series of deletion and substitution constructs in transgenic potato and tobacco plants. In a construct containing 3.9 kb of 5' flanking region, substitution of the native 3' sequence with the nopaline synthase 3' sequence and deletion of the leader intron did not significantly affect expression in vegetative tissues. However, in a construct containing only 320 bp of 5' flanking region, these changes had marked effects. Replacing the native 3' sequences with nopaline synthase 3' sequences caused a six- to 20-fold increase in expression in vascular tissue, and removing the leader intron almost completely abolished expression in potato plants. Surprisingly, removal of the leader intron from either the full-length construct or a construct containing only 320 bp of 5' flanking sequence reduced expression in vascular tissue of tobacco anthers at later stages of development but increased expression in pollen by more than 100-fold.     

Several hundred base pairs upstream of Drosophila hsp23 and 26 genes are required for their heat induction in transformed flies.

We have used the P-element-mediated transformation of Drosophila germ line to study the 5' DNA sequences involved in the thermal inducibility of the genes for heat shock proteins hsp23 and 26. The results are strikingly different from those previously obtained in heterologous systems. For hsp23, each successive shortening of the promoter region from 618 to 402, 321 and 263 bp clearly decreased the expression. A construct with only 149 bp was not inducible at all. For hsp26, all the regulatory elements appear to be clustered in the first 350 bp upstream from the cap site. Clones with 171 bp showed a 4- to 10-fold decrease in induction depending on the transformed line, and those with only 52 bp were not expressed. The results suggest that at least three Pelham consensus sequences are required for the full expression of these two genes. The direct involvement of one of these consensus sequences has been assessed: a 6-bp deletion within the proximal element of the hsp26 gene strongly reduced its inducibility. Our results also indicate that X-linked hsp genes exhibit either partial dosage compensation or none at all.