Ecdysterone regulatory elements function as both transcriptional activators and repressors

1991 ◽  
Vol 11 (4) ◽  
pp. 1846-1853
Author(s):  
L Dobens ◽  
K Rudolph ◽  
E M Berger
Keyword(s):  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Transcriptional Activators ◽  
Reporter Plasmid ◽  
Bacterial Gene ◽  
Simplex Virus ◽  
Very High ◽  
Cat Gene ◽  
Cat Expression

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.

scholarly journals Ecdysterone regulatory elements function as both transcriptional activators and repressors.

1991 ◽  
Vol 11 (4) ◽  
pp. 1846-1853 ◽  
Author(s):  
L Dobens ◽  
K Rudolph ◽  
E M Berger
Keyword(s):  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Transcriptional Activators ◽  
Reporter Plasmid ◽  
Bacterial Gene ◽  
Simplex Virus ◽  
Very High ◽  
Cat Gene ◽  
Cat Expression

A synthetic, 23-bp ecdysterone regulatory element (EcRE), derived from the upstream region of the Drosophila melanogaster hsp27 gene, was inserted adjacent to the herpes simplex virus thymidine kinase promoter fused to a bacterial gene for chloramphenicol acetyltransferase (CAT). Hybrid constructs were transfected into Drosophila S3 cells and assayed for ecdysterone-inducible CAT expression. In the absence of ecdysterone a tandem pair of EcREs repressed the high constitutive level of CAT activity found after transfection with the parent reporter plasmid alone. After hormone addition very high levels of CAT activity were observed. Insertion of the EcRE pair 3' of the CAT gene also led to high levels of ecdysterone-induced CAT expression, but the repression of high constitutive levels of CAT activity failed to occur. The EcRE-CAT construct was cotransfected with plasmids containing tandem 10-mers or 40-mers of the EcRE but lacking a reporter gene. These additional EcREs led to a reduced level of ecdysterone-induced CAT activity and to an elevation of basal CAT activity in the absence of hormone. The data suggest that the receptor binds to the EcRE in the absence of hormone, blocking basal transcription from a constitutive promoter. In the presence of ecdysterone, receptor-hormone binding to the EcRE leads to greatly enhanced transcription.

scholarly journals Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements.

1996 ◽  
Vol 16 (5) ◽  
pp. 2408-2417 ◽  
Author(s):  
M Nakayama ◽  
J Stauffer ◽  
J Cheng ◽  
S Banerjee-Basu ◽  
E Wawrousek ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Spatial Organization ◽  
Molecular Mechanisms ◽  
Fiber Type ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Regulatory Sequences ◽  
Fast Fiber ◽  
Core Elements

The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb -0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5-kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles, indicating that these regulatory elements are sufficient to confer fiber-type-specific transcription. Alignment of rat TnIs and quail TnIf regulatory sequences indicates that there is a conserved spatial organization of core elements, namely, an E box, a CCAC box, a MEF-2-like sequence, and a previously uncharacterized motif. The core elements were shown to bind their cognate factors by electrophoretic mobility shift assays, and their mutation demonstrated that the TnIs CCAC and E boxes are necessary for transgene expression. Our results suggest that the interaction of closely related transcriptional protein-DNA complexes is utilized to specify fiber type diversity.

scholarly journals Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia.

1987 ◽  
Vol 7 (5) ◽  
pp. 1807-1814 ◽  
Author(s):  
A B Chepelinsky ◽  
B Sommer ◽  
J Piatigorsky
Keyword(s):  
Dna Sequences ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Promoter Sequences ◽  
Hybrid Genes ◽  
Cat Gene ◽  
Cat Expression

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

Interaction between two different regulatory elements activates the murine alpha A-crystallin gene promoter in explanted lens epithelia

1987 ◽  
Vol 7 (5) ◽  
pp. 1807-1814
Author(s):  
A B Chepelinsky ◽  
B Sommer ◽  
J Piatigorsky
Keyword(s):  
Dna Sequences ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Hybrid Gene ◽  
Promoter Sequences ◽  
Hybrid Genes ◽  
Cat Gene ◽  
Cat Expression

Previous experiments have indicated that 5' flanking DNA sequences (nucleotides-366 to +46) are capable of regulating the lens-specific transcription of the murine alpha A-crystallin gene. Here we have analyzed these 5' regulatory sequences by transfecting explanted embryonic chicken lens epithelia with different alpha A-crystallin-CAT (chloramphenicol acetyltransferase) hybrid genes (alpha A-crystallin promoter sequences fused to the bacterial CAT gene in the pSVO-CAT expression vector). The results indicated the presence of a proximal (-88 to +46) and a distal (-111 to -88) domain which must interact for promoter function. Deletion experiments showed that the sequence between -88 and -60 was essential for function of the proximal domain in the explanted epithelia. A synthetic oligonucleotide containing the sequence between -111 and -84 activated the proximal domain when placed in either orientation 57 base pairs upstream from position -88 of the alpha A-crystallin-CAT hybrid gene.

scholarly journals Positive and negative regulatory elements in the upstream region of the rat Cu/Zn-superoxide dismutase gene

1999 ◽  
Vol 339 (2) ◽  
pp. 335-341 ◽  
Author(s):  
Mun Seog CHANG ◽  
Hae Yong YOO ◽  
Hyune Mo RHO
Keyword(s):  
Superoxide Dismutase ◽  
Transcription Factors ◽  
Binding Sites ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Sod1 Gene ◽  
Mobility Shift ◽  
Heterologous Promoter ◽  
Natural Context

Cu/Zn-superoxide dismutase (SOD1) catalyses the dismutation of superoxide radicals and neutralizes the oxidative effects of various chemicals. Deletion analysis of the upstream region of the rat SOD1 gene revealed that the promoter contains a positive regulatory element (PRE) and a negative regulatory element (NRE), which encompass the regions from -576 to -412 and from -412 to -305 respectively from the site of initiation of transcription. These DNA elements showed enhancer and silencer activities respectively in the natural context and in a heterologous promoter system. Using an electrophoretic-mobility-shift assay and a supershift assay with a specific antibody, the cis-elements of the PRE and NRE were identified as binding sites for transcription factors Elk1 and YY1 (Ying-Yang 1) respectively. Consistent with the presumed roles of the PRE and NRE, Elk1 increased SOD1 gene transcription about 4–5-fold, whereas YY1 exerted a negative effect of about 6-fold. Mutations of the Elk1- and YY1-binding sites led to diminution and elevation respectively of transcriptional activities, both in the natural context and in heterologous promoter systems. These results suggest that the transcription factors Elk1 and YY1, binding in the PRE and NRE respectively, co-ordinate the expression of the SOD1 gene.

scholarly journals S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene.

1989 ◽  
Vol 9 (3) ◽  
pp. 1005-1013 ◽  
Author(s):  
I Hwang ◽  
C B Chae
Keyword(s):  
Dna Synthesis ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
S Phase ◽  
Upstream Region ◽  
Base Pairs ◽  
Regulated Expression ◽  
Dna Fragment ◽  
Pachytene Spermatocytes

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.

scholarly journals cis-acting regulatory elements within gag genes of avian retroviruses.

1987 ◽  
Vol 7 (1) ◽  
pp. 388-397 ◽  
Author(s):  
S Arrigo ◽  
M Yun ◽  
K Beemon
Keyword(s):  
Rous Sarcoma Virus ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Enhancer Element ◽  
Cis Acting ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Cat Gene ◽  
Internal Fragment ◽  
Cat Expression

A cis-acting enhancer element has been detected within the gag gene of several avian retroviruses, including Rous sarcoma virus, Fujinami sarcoma virus, and the endogenous Rous-associated virus-0. A consensus enhancer core sequence, GTGGTTTG, is present in all of these viral genomes, approximately 900 bases downstream from the site of initiation of transcription. When an internal fragment derived from the gag gene of any of these viruses (spanning nucleotides 533 to approximately 1149) was inserted into a plasmid containing the chloramphenicol acetyltransferase (cat) gene under control of the simian virus 40 promoter, 9- or 21-fold enhancement of CAT expression was observed after transfection into mouse L cells and chicken embryo fibroblasts, respectively. This enhancement was not dependent on the position of insertion of the gag fragment into the plasmid. However, there was a strong dependence on orientation, with higher levels of CAT expression in constructs in which the 5' end of the gag fragment was nearest to the promoter, suggesting a possible negative regulatory element at the 3' end of this fragment. Deletion of the 3' end of the insert resulted in a gag fragment, containing nucleotides 533 to 1017, which enhanced expression equally in either orientation. When the gag fragment was inserted into a plasmid containing the cat gene under the control of an intact Rous sarcoma virus long terminal repeat, it induced a two- to threefold increase in CAT activity and CAT mRNA levels. Translation of the gag fragment did not appear to be necessary for the observed enhancement, since two insertional mutations resulting in frameshifts in the gag insert did not affect CAT expression. However, deletion of a 330-base internal fragment from the gag insert restored a basal level of CAT activity. These results suggest that retroviruses have regulatory elements within their genes distinct from those in the long terminal repeats that flank the genes.

cis-acting regulatory elements within gag genes of avian retroviruses

1987 ◽  
Vol 7 (1) ◽  
pp. 388-397
Author(s):  
S Arrigo ◽  
M Yun ◽  
K Beemon
Keyword(s):  
Rous Sarcoma Virus ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Enhancer Element ◽  
Cis Acting ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Cat Gene ◽  
Internal Fragment ◽  
Cat Expression

A cis-acting enhancer element has been detected within the gag gene of several avian retroviruses, including Rous sarcoma virus, Fujinami sarcoma virus, and the endogenous Rous-associated virus-0. A consensus enhancer core sequence, GTGGTTTG, is present in all of these viral genomes, approximately 900 bases downstream from the site of initiation of transcription. When an internal fragment derived from the gag gene of any of these viruses (spanning nucleotides 533 to approximately 1149) was inserted into a plasmid containing the chloramphenicol acetyltransferase (cat) gene under control of the simian virus 40 promoter, 9- or 21-fold enhancement of CAT expression was observed after transfection into mouse L cells and chicken embryo fibroblasts, respectively. This enhancement was not dependent on the position of insertion of the gag fragment into the plasmid. However, there was a strong dependence on orientation, with higher levels of CAT expression in constructs in which the 5' end of the gag fragment was nearest to the promoter, suggesting a possible negative regulatory element at the 3' end of this fragment. Deletion of the 3' end of the insert resulted in a gag fragment, containing nucleotides 533 to 1017, which enhanced expression equally in either orientation. When the gag fragment was inserted into a plasmid containing the cat gene under the control of an intact Rous sarcoma virus long terminal repeat, it induced a two- to threefold increase in CAT activity and CAT mRNA levels. Translation of the gag fragment did not appear to be necessary for the observed enhancement, since two insertional mutations resulting in frameshifts in the gag insert did not affect CAT expression. However, deletion of a 330-base internal fragment from the gag insert restored a basal level of CAT activity. These results suggest that retroviruses have regulatory elements within their genes distinct from those in the long terminal repeats that flank the genes.

scholarly journals S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene

1989 ◽  
Vol 9 (3) ◽  
pp. 1005-1013
Author(s):  
I Hwang ◽  
C B Chae
Keyword(s):  
Dna Synthesis ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
S Phase ◽  
Upstream Region ◽  
Base Pairs ◽  
Regulated Expression ◽  
Dna Fragment ◽  
Pachytene Spermatocytes

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.

Transvection and Silencing of the Scr Homeotic Gene of Drosophila melanogaster

Genetics ◽  
2002 ◽  
Vol 161 (2) ◽  
pp. 733-746
Author(s):  
Jeffrey W Southworth ◽  
James A Kennison
Keyword(s):  
Drosophila Melanogaster ◽  
Chromosomal Aberrations ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Anomalous Behavior ◽  
Polycomb Group ◽  
Homeotic Gene ◽  
Polycomb Group Proteins ◽  
Sex Combs Reduced ◽  
In Cis

Abstract The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.

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