Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids

1985 ◽  
Vol 5 (8) ◽  
pp. 2070-2079
Author(s):  
F G Kern ◽  
L Dailey ◽  
C Basilico
Keyword(s):  
Gene Expression ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Coding Region ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Enhancer Sequence ◽  
In Cells

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

scholarly journals Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

1985 ◽  
Vol 5 (8) ◽  
pp. 2070-2079 ◽  
Author(s):  
F G Kern ◽  
L Dailey ◽  
C Basilico
Keyword(s):  
Gene Expression ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Simian Virus 40 ◽  
Regulatory Elements ◽  
Coding Region ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Enhancer Sequence ◽  
In Cells

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

scholarly journals Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

2005 ◽  
Vol 79 (8) ◽  
pp. 4918-4926 ◽  
Author(s):  
Kathryn M. Spink ◽  
Laimonis A. Laimins
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Viral Genome ◽  
Copy Number ◽  
Dna Amplification ◽  
Regulatory Elements ◽  
Late Gene ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Viral Copy Number

ABSTRACT The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine β-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.

scholarly journals Chromatin accessibility analysis uncovers regulatory element landscape in prostate cancer progression

2020 ◽  
Author(s):  
Joonas Uusi-Mäkelä ◽  
Ebrahim Afyounian ◽  
Francesco Tabaro ◽  
Tomi Häkkinen ◽  
Alessandro Lussana ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Cancer Progression ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Prostate Cancer Progression ◽  
Accessibility Analysis ◽  
A Genome

AbstractAberrant oncogene functions and structural variation alter the chromatin structure in cancer cells. While gene regulation by chromatin states has been studied extensively, chromatin accessibility and its relevance in aberrant gene expression during prostate cancer progression is not well understood. Here, we report a genome-wide chromatin accessibility analysis of clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration-resistant prostate cancer (CRPC) and integrative analysis with transcriptome, methylome, and proteome profiles of the same samples to uncover disease-relevant regulatory elements and their association to altered gene expression during prostate cancer progression. While promoter accessibility is consistent during disease initiation and progression, at distal sites chromatin accessibility is variable enabling transcription factors (TFs) binding patterns that are differently activated in different patients and disease stages. We identify consistent progression-related chromatin alterations during the progression to CRPC. By studying the TF binding patterns, we demonstrate the activation and suppression of androgen receptor-driven regulatory programs during PC progression and identify complementary TF regulatory modules characterized by e.g. MYC and glucocorticoid receptor. By correlation analysis we assign at least one putative regulatory region for 62% of genes and 85% of proteins differentially expressed during prostate cancer progression. Taken together, our analysis of the chromatin landscape in PC identifies putative regulatory elements for the majority of cancer-associated genes and characterizes their impact on the cancer phenotype.

scholarly journals Human papillomavirus type 16 late gene expression is regulated by cellular RNA processing factors in response to epithelial differentiation

2008 ◽  
Vol 36 (3) ◽  
pp. 522-524 ◽  
Author(s):  
Sarah A. Cumming ◽  
Thanaporn Cheun-Im ◽  
Stephen G. Milligan ◽  
Sheila V. Graham
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
Current Knowledge ◽  
Regulatory Element ◽  
Epithelial Differentiation ◽  
Late Gene ◽  
Coding Region ◽  
Late Gene Expression ◽  
Human Papillomavirus Type 16 ◽  
L1 And L2

HPV16 (human papillomavirus type 16) is a 7.9 kb double-stranded DNA virus that infects anogenital mucosal epithelia. In some rare cases, in women, infection can progress to cervical cancer. HPV16 gene expression is regulated through use of multiple promoters and alternative splicing and polyadenylation. The virus genome can be divided into an early and a late coding region. The late coding region contains the L1 and L2 genes. These encode the virus capsid proteins L1 and L2; protein expression is confined to the upper epithelial layers and is regulated post-transcriptionally in response to epithelial differentiation. A 79 nt RNA regulatory element, the LRE (late regulatory element), involved in this regulation is sited at the 3′-end of the L1 gene and extends into the late 3′-UTR (3′-untranslated region). This element represses late gene expression in differentiated epithelial cells and may activate it in differentiated cells. The present paper describes our current knowledge of LRE RNA–protein interaction and their possible functions.

scholarly journals The Human Papillomavirus Type 31 Late 3′ Untranslated Region Contains a Complex Bipartite Negative Regulatory Element

2002 ◽  
Vol 76 (12) ◽  
pp. 5993-6003 ◽  
Author(s):  
Sarah A. Cumming ◽  
Claire E. Repellin ◽  
Maria McPhillips ◽  
Jonathan C. Radford ◽  
J. Barklie Clements ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Papillomavirus ◽  
High Risk ◽  
Epithelial Cells ◽  
Regulatory Element ◽  
Capsid Proteins ◽  
Cellular Proteins ◽  
Late Gene Expression ◽  
Hpv 16 ◽  
Negative Regulatory Element

ABSTRACT The papillomavirus life cycle is tightly linked to epithelial cell differentiation. Production of virus capsid proteins is restricted to the most terminally differentiated keratinocytes in the upper layers of the epithelium. However, mRNAs encoding the capsid proteins can be detected in less-differentiated cells, suggesting that late gene expression is controlled posttranscriptionally. Short sequence elements (less than 80 nucleotides in length) that inhibit gene expression in undifferentiated epithelial cells have been identified in the late 3′ untranslated regions (UTRs) of several papillomaviruses, including the high-risk mucosal type human papillomavirus type 16 (HPV-16). Here we show that closely related high-risk mucosal type HPV-31 also contains elements that can act to repress gene expression in undifferentiated epithelial cells. However, the HPV-31 negative regulatory element is surprisingly complex, comprising a major inhibitory element of approximately 130 nucleotides upstream of the late polyadenylation site and a minor element of approximately 110 nucleotides mapping downstream. The first 60 nucleotides of the major element have 68% identity to the negative regulatory element of HPV-16, and these elements bind the same cellular proteins, CstF-64, U2AF65, and HuR. The minor inhibitory element binds some cellular proteins in common with the major inhibitory element, though it also binds certain proteins that do not bind the upstream element.

Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2896-2909 ◽  
Author(s):  
E A Sternberg ◽  
G Spizz ◽  
W M Perry ◽  
D Vizard ◽  
T Weil ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Muscle Cells ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Simian Virus ◽  
Cell Type ◽  
Specific Expression ◽  
Developing Muscle

Terminal differentiation of skeletal myoblasts is accompanied by induction of a series of tissue-specific gene products, which includes the muscle isoenzyme of creatine kinase (MCK). To begin to define the sequences and signals involved in MCK regulation in developing muscle cells, the mouse MCK gene has been isolated. Sequence analysis of 4,147 bases of DNA surrounding the transcription initiation site revealed several interesting structural features, some of which are common to other muscle-specific genes and to cellular and viral enhancers. To test for sequences required for regulated expression, a region upstream of the MCK gene from -4800 to +1 base pairs, relative to the transcription initiation site, was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. Introduction of this MCK-CAT fusion gene into C2 muscle cells resulted in high-level expression of CAT activity in differentiated myotubes and no detectable expression in proliferating undifferentiated myoblasts or in nonmyogenic cell lines. Deletion mutagenesis of sequences between -4800 and the transcription start site showed that the region between -1351 and -1050 was sufficient to confer cell type-specific and developmentally regulated expression on the MCK promoter. This upstream regulatory element functioned independently of position, orientation, or distance from the promoter and therefore exhibited the properties of a classical enhancer. This upstream enhancer also was able to confer muscle-specific regulation on the simian virus 40 promoter, although it exhibited a 3- to 5-fold preference for its own promoter. In contrast to the cell type- and differentiation-specific expression of the upstream enhancer, the MCK promoter was able to function in myoblasts and myotubes and in nonmyogenic cell lines when combined with the simian virus 40 enhancer. An additional positive regulatory element was identified within the first intron of the MCK gene. Like the upstream enhancer, this intragenic element functioned independently of position, orientation, and distance with respect to the MCK promoter and was active in differentiated myotubes but not in myoblasts. These results demonstrate that expression of the MCK gene in developing muscle cells is controlled by complex interactions among multiple upstream and intragenic regulatory elements that are functional only in the appropriate cellular context.

Downstream sequences affect transcription initiation from the adenovirus major late promoter

1986 ◽  
Vol 6 (7) ◽  
pp. 2684-2694 ◽  
Author(s):  
S L Mansour ◽  
T Grodzicker ◽  
R Tjian
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Control Element ◽  
Coding Region ◽  
Late Promoter ◽  
Sv40 T Antigen ◽  
Transient Expression Assay ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and +190 by showing that insertion of SV40 sequences within the intron after the first leader segment at +190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.

Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen

Development ◽  
1998 ◽  
Vol 125 (21) ◽  
pp. 4185-4193 ◽  
Author(s):  
Q. Gao ◽  
R. Finkelstein
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Repeated Sequence ◽  
Drosophila Embryo ◽  
Sequence Motif ◽  
Specific Expression ◽  
Gap Gene

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

Identification of a regulatory region that mediates glucose-dependent induction of the Saccharomyces cerevisiae enolase gene ENO2

1986 ◽  
Vol 6 (7) ◽  
pp. 2287-2297
Author(s):  
R Cohen ◽  
J P Holland ◽  
T Yokoi ◽  
M J Holland
Keyword(s):  
Gene Expression ◽  
Regulatory Region ◽  
Carbon Sources ◽  
Initiation Site ◽  
Deletion Mapping ◽  
Regulatory Regions ◽  
Coding Sequences ◽  
Flanking Region ◽  
Fused Gene ◽  
In Cells

There are two yeast enolase genes, designated ENO1 and ENO2, which are expressed differentially in vegetative cells grown on glucose and in cells grown on gluconeogenic carbon sources. ENO2 is induced more than 20-fold in cells grown on glucose, whereas ENO1 expression is similar in cells grown on glucose and in cells grown on gluconeogenic carbon sources. Sequences within the 5' flanking region of ENO2 which are required for glucose-dependent induction were identified by deletion mapping analysis. These studies were carried out by using a fused gene containing the ENO2 5' flanking sequences and the ENO1 coding sequences. This fused gene undergoes glucose-dependent induction and is expressed at the same level as the resident ENO2 gene in cells grown on glucose or gluconeogenic carbon sources. Expression of fused genes containing deletion mutations within the ENO2 5' flanking region was monitored after integration at the ENO1 locus of a strain carrying a deletion of the resident ENO1 coding sequences. This analysis showed that there are two upstream activation sites located immediately upstream and downstream from a position 461 base pairs upstream from the transcriptional initiation site. Either one of these upstream activation sites is sufficient for glucose-dependent induction and normal gene expression in the presence of gluconeogenic carbon sources. Deletion of both regulatory regions results in a complete loss of gene expression. The regulatory regions function normally in both orientations relative to the coding sequences. Mutant fused genes containing small deletions within the regulatory regions were constructed; these genes were expressed normally in gluconeogenic carbon sources but were not induced in the presence of glucose. Based on this analysis, ENO2 contains a cis-acting regulatory region which is required for gene expression and mediates glucose-dependent induction of gene expression.

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