Negative Regulatory Sequences Research Articles

Identification of an inducible factor that binds to a positive regulatory element of the human beta-interferon gene.

Human beta-interferon gene expression is induced by virus or poly(I).poly(C). This induction is due at least in part to an increase in the rate of transcription and does not require protein synthesis. A 40-base-pair DNA sequence within the beta-interferon promoter, termed the interferon gene regulatory element (IRE), is an inducible enhancer in mouse fibroblasts, and both positive and negative regulatory DNA sequences have been identified within this element. In this paper we identify three factors that bind specifically to two positive regulatory domains within the IRE. Two of these factors are present in nuclear extracts prepared from uninduced and induced cells; one is present only in extracts from induced cells. The functional significance of these binding activities was demonstrated by showing that point mutations within the IRE that decrease human beta-interferon gene transcription in vivo prevent binding in vitro. We propose that induction of the beta-interferon gene involves the modification of a protein to a form that binds specifically to a positive regulatory sequence within the IRE.

Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus.

The art gene product of the human immunodeficiency virus is required for the expression of virion capsid and envelope gene proteins. The experiments presented here show that sequences located within the coding region of the envelope gene exert a negative effect on the expression of heterologous genes and that the negative effect of these sequences can be relieved by the art gene product. This region in the env gene contains negative regulatory sequences that inhibit gene expression, as well as a sequence necessary for the art gene product-dependent relief of repression. The experiments define the cis- and trans-acting components of a regulatory system that permits differential expression of human immunodeficiency virus virion structural and regulatory proteins.

The Fos complex and Fos-related antigens recognize sequence elements that contain AP-1 binding sites.

The Fos protein complex and several Fos-related antigens bind directly or indirectly to a common sequence element that is similar to the consensus binding site for HeLa cell activator protein 1 (AP-1). This element is present in a negative regulatory sequence in the differentiation-sensitive adipocyte gene, aP2; in a transcriptional enhancer for the Gibbon ape leukemia virus; and in a region of the human immunodeficiency virus (HIV) long terminal repeat partially characterized as a negative regulatory element. The protein level and binding activity of Fos and Fos-related antigens increase rapidly after calcium ionophore treatment of a CD4+ human lymphoblast cell line, H9. These data suggest that several proteins may associate with the AP-1 binding site. Moreover, temporally regulated control of the level of each protein could represent a mechanism for modulation of these putative mediators of gene expression.

Overlapping positive and negative regulatory domains of the human beta-interferon gene.

Virus or poly(I).poly(C) induction of human beta-interferon gene expression requires a 40-base-pair DNA sequence designated the interferon gene regulatory element (IRE). Previous studies have shown that the IRE contains both positive and negative regulatory DNA sequences. To localize these sequences and study their interactions, we have examined the effects of a large number of single-base mutations within the IRE on beta-interferon gene regulation. We find that the IRE consists of two genetically separable positive regulatory domains and an overlapping negative control sequence. We propose that the beta-interferon gene is switched off in uninduced cells by a repressor that blocks the interaction between one of the two positive regulatory sequences and a specific transcription factor. Induction would then lead to inactivation or displacement of the repressor and binding of transcription factors to both positive regulatory domains.

Use of a stable bovine papillomavirus vector to study inducible genes.

The human beta interferon (hIFN beta 1) gene has been cloned into a bovine papillomavirus type 1 (BPV-1)/pML2 vector. The recombinant DNAs can be maintained as stable plasmids in mouse cells, with no detectable rearrangements. The linked hIFN beta 1 gene is inducible with little line-to-line variability in independently transformed cell lines. The hIFN beta 1 gene is equally induced when it is inserted adjacent to or away from the BPV-1 enhancer. The great stability of the BPV-1/pML2 vector has allowed us to study the regulation of the hIFN beta 1 gene in mouse cells, with the construction of deleted and hybrid hIFN beta 1 genes. Using this strategy, we have confirmed that the 5' sequences of the hIFN beta 1 gene are involved in the induction mechanism. The entire hIFN beta 1 regulatory element is able to express heterologous genes with no induction, suggesting that the negative regulatory sequences present in the 3' region of the hIFN beta 1 regulatory element are not fully active on heterologous genes.

Transcription of the constitutively expressed yeast enolase gene ENO1 is mediated by positive and negative cis-acting regulatory sequences.

There are two enolase genes, ENO1 and ENO2, per haploid yeast genome. Expression of the ENO1 gene is quantitatively similar in cells grown on glucose or gluconeogenic carbon sources. In contrast, ENO2 expression is induced more than 20-fold in cells grown on glucose as the carbon source. cis-Acting regulatory sequences were mapped within the 5'-flanking region of the constitutively expressed yeast enolase gene ENO1. A complex positive regulatory region was located 445 base pairs (bp) upstream from the transcriptional initiation site which was required for ENO1 expression in cells grown on glycolytic or gluconeogenic carbon sources. A negative regulatory region was located 160 bp upstream from the transcriptional initiation site. Sequences required for the function of this negative regulatory element were mapped to a 38-bp region. Deletion of all or a portion of these latter sequences permitted glucose-dependent induction of ENO1 expression that was quantitatively similar to that of the glucose-inducible ENO2 gene. The negative regulatory element therefore prevents glucose-dependent induction of the ENO1 gene. Hybrid 5'-flanking regions were constructed which contained the upstream regulatory sequences of one enolase gene fused at a site upstream from the TATAAA box in the other enolase gene. Analysis of the expression of enolase genes containing these hybrid 5'-flanking region showed that the positive regulatory regions of ENO1 and ENO2 were functionally similar, as were the regions extending from the TATAAA boxes to the initiation codons. Based on these studies, we conclude that the negative regulatory element plays the critical role in maintaining the constitutive expression of the ENO1 structural gene in cells grown on glucose or gluconeogenic carbon sources.

Transcription of the constitutively expressed yeast enolase gene ENO1 is mediated by positive and negative cis-acting regulatory sequences.

There are two enolase genes, ENO1 and ENO2, per haploid yeast genome. Expression of the ENO1 gene is quantitatively similar in cells grown on glucose or gluconeogenic carbon sources. In contrast, ENO2 expression is induced more than 20-fold in cells grown on glucose as the carbon source. cis-Acting regulatory sequences were mapped within the 5'-flanking region of the constitutively expressed yeast enolase gene ENO1. A complex positive regulatory region was located 445 base pairs (bp) upstream from the transcriptional initiation site which was required for ENO1 expression in cells grown on glycolytic or gluconeogenic carbon sources. A negative regulatory region was located 160 bp upstream from the transcriptional initiation site. Sequences required for the function of this negative regulatory element were mapped to a 38-bp region. Deletion of all or a portion of these latter sequences permitted glucose-dependent induction of ENO1 expression that was quantitatively similar to that of the glucose-inducible ENO2 gene. The negative regulatory element therefore prevents glucose-dependent induction of the ENO1 gene. Hybrid 5'-flanking regions were constructed which contained the upstream regulatory sequences of one enolase gene fused at a site upstream from the TATAAA box in the other enolase gene. Analysis of the expression of enolase genes containing these hybrid 5'-flanking region showed that the positive regulatory regions of ENO1 and ENO2 were functionally similar, as were the regions extending from the TATAAA boxes to the initiation codons. Based on these studies, we conclude that the negative regulatory element plays the critical role in maintaining the constitutive expression of the ENO1 structural gene in cells grown on glucose or gluconeogenic carbon sources.

Analysis of the transforming potential of the human homolog of mos

The human homology, c- mos hu, of the mouse cellular mos proto-oncogene (c- mos mu) transforms NIH 3T3 cells at low efficiency. Furthermore, the c- mos hu-induced foci are less distinct, and transformed cells contain a high level of human mos protein. The transforming activity of hybrid mos genes derived from human and mouse sequences reveals three domains within the coding region, as well as a negative regulatory sequence upstream from the c- mos hu ORF that reduces its transforming efficiency. The mos C-terminal region, however, which contains the src-kinase homology domain, appears to have the greatest influence on transforming efficiency. The low transforming efficiency of c- mos hu may provide a selective advantage to the host, but it also may indicate a reduced or modified function of mos in humans.

The human β-interferon gene enhancer is under negative control

The human β-interferon gene is regulated by an inducible enhancer element. Analysis of the effect of deletions within this element on β-interferon transcription indicates that this enhancer is under negative control. Deletion of sequences from the 3′ end of the enhancer leads to a dramatic increase in the basal level of β-interferon mRNA and a decrease in the induction ratio. The remaining 5′ region of the enhancer can act as a strong constitutive transcription element, and it shares considerable homology with sequences known to be required for the activity of constitutive viral enhancers. We conclude that the β-interferon enhancer consists of a constitutive transcription element and a negative regulatory sequence that prevents enhancer activity prior to induction. Thus, derepression of a constitutive transcription element appears to play a key role in the control of human β-interferon gene expression.

A negative regulatory sequence near the mouse beta-maj globin gene associated with a region of potential Z-DNA.

The possible regulatory role of DNA sequences situated 5' to the beta-maj globin gene was investigated by two types of assay. First, a long term transformation assay was used to measure the efficiency of transformation of TK- mouse (LATK-) and hamster (BHKTK-) fibroblast cells with DNA molecules made by covalently linking mouse and human DNA fragments to the herpes simplex virus (HSV-1) thymidine kinase (tk) gene in the plasmid pTK1. When the promoter regions from the mouse beta-maj globin or the human epsilon-globin genes are substituted for the viral promoter in the tk gene transformation occurs with 10-20% of the efficiency of the original plasmid. A fragment (H1), containing sequences between 344 and 1413 bp upstream from the mouse beta-maj globin cap site, almost completely abolishes transformation when inserted next to hybrid tk genes containing the mouse beta-globin or human epsilon-globin promoter but has no effect on the intact tk gene. The effect can be demonstrated with the H1 fragment in either orientation relative to the tk gene. Secondly, in transient expression assays the H1 fragment strongly inhibits transcription when covalently linked to the tk gene under control of either globin promoter, but not when linked to the tk gene with its own promoter. The H1 fragment contains 53 bp of purine-pyrimidine alternation (ACAT)n as part of a larger region potentially capable of adopting a Z-DNA conformation.