Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites

1986 ◽  
Vol 6 (2) ◽  
pp. 452-461 ◽  
Author(s):  
J A Nelson ◽  
M Groudine
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Features ◽  
In Vitro Transcription ◽  
Dnase I ◽  
Early Gene ◽  
Immediate Early ◽  
Dnase I Hypersensitive Sites ◽  
Teratocarcinoma Cells ◽  
Hypersensitive Sites

Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.

scholarly journals Transcriptional regulation of the human cytomegalovirus major immediate-early gene is associated with induction of DNase I-hypersensitive sites.

1986 ◽  
Vol 6 (2) ◽  
pp. 452-461 ◽  
Author(s):  
J A Nelson ◽  
M Groudine
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Features ◽  
In Vitro Transcription ◽  
Dnase I ◽  
Early Gene ◽  
Immediate Early ◽  
Dnase I Hypersensitive Sites ◽  
Teratocarcinoma Cells ◽  
Hypersensitive Sites

Human teratocarcinoma cells were used to examine structural features associated with expression of the major immediate-early (IE) gene of human cytomegalovirus. By immunofluorescence, comparison of RNA levels, and in vitro transcription of nuclei, we showed that the major IE gene is inactive in undifferentiated but active in differentiated cells. Therefore, the block in human cytomegalovirus replication in teratocarcinoma cells appears to be at the transcriptional level, in one of the initial genes transcribed. In addition, the in vitro transcription experiments demonstrated that in permissive infections the gene was transcriptionally inactive late in infection. A comparison of the structural features of the promoter region with the active and inactive IE genes showed the presence of constitutive and inducible DNase I-hypersensitive sites. The majority of the constitutive sites existed at -175, -275, -375, -425, and -525 relative to the cap site in an area which has been shown to be capable of simian virus 40 enhancer function. In contrast, the inducible DNase I sites were located outside this region at -650, -775, -875, and -975.

scholarly journals Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene.

1987 ◽  
Vol 7 (11) ◽  
pp. 4125-4129 ◽  
Author(s):  
J A Nelson ◽  
C Reynolds-Kohler ◽  
B A Smith
Keyword(s):  
Human Cytomegalovirus ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early Gene ◽  
Dnase I ◽  
Early Gene ◽  
Immediate Early ◽  
Flanking Sequence ◽  
Hypersensitive Sites

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.

Negative and positive regulation by a short segment in the 5'-flanking region of the human cytomegalovirus major immediate-early gene

1987 ◽  
Vol 7 (11) ◽  
pp. 4125-4129
Author(s):  
J A Nelson ◽  
C Reynolds-Kohler ◽  
B A Smith
Keyword(s):  
Human Cytomegalovirus ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Fold Increase ◽  
Immediate Early Gene ◽  
Dnase I ◽  
Early Gene ◽  
Immediate Early ◽  
Flanking Sequence ◽  
Hypersensitive Sites

To analyze the significance of inducible DNase I-hypersensitive sites occurring in the 5'-flanking sequence of the major immediate-early gene of human cytomegalovirus (HCMV), various deleted portions of the HCMV immediate-early promoter regulatory region were attached to the chloramphenicol acetyltransferase (CAT) gene and assayed for activity in transiently transfected undifferentiated and differentiated human teratocarcinoma cells, Tera-2. Assays of progressive deletions in the promoter regulatory region indicated that removal of a 395-base-pair portion of this element (nucleotides -750 to -1145) containing two inducible DNase I sites which correlate with gene expression resulted in a 7.5-fold increase in CAT activity in undifferentiated cells. However, in permissive differentiated Tera-2, human foreskin fibroblast, and HeLa cells, removal of this regulatory region resulted in decreased activity. In addition, attachment of this HCMV upstream element to a homologous or heterologous promoter increased activity three- to fivefold in permissive cells. Therefore, a cis regulatory element exists 5' to the enhancer of the major immediate-early gene of HCMV. This element negative modulates expression in nonpermissive cells but positively influences expression in permissive cells.

scholarly journals In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins.

1993 ◽  
Vol 13 (2) ◽  
pp. 1238-1250 ◽  
Author(s):  
K M Klucher ◽  
M Sommer ◽  
J T Kadonaga ◽  
D H Spector
Keyword(s):  
Human Cytomegalovirus ◽  
Hela Cells ◽  
Transcriptional Activation ◽  
Histone H1 ◽  
Early Gene ◽  
Immediate Early ◽  
Early Promoter ◽  
Drosophila Embryos

To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro. In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels. In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins. To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos. The results of the in vitro experiments correlated well with those obtained in vivo. The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein. With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein. Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter. However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold. The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Krüppel promoter in either the presence or absence of histone H1. These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

scholarly journals Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor.

1991 ◽  
Vol 11 (9) ◽  
pp. 4314-4323 ◽  
Author(s):  
J Hapgood ◽  
S Cuthill ◽  
P Söderkvist ◽  
A Wilhelmsson ◽  
I Pongratz ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Nuclear Extract ◽  
Initiation Site ◽  
Positive Control ◽  
Dnase I ◽  
Response Elements ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Dioxin Receptor

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand beta-naphthoflavone. In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.

Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor

1991 ◽  
Vol 11 (9) ◽  
pp. 4314-4323
Author(s):  
J Hapgood ◽  
S Cuthill ◽  
P Söderkvist ◽  
A Wilhelmsson ◽  
I Pongratz ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Nuclear Extract ◽  
Initiation Site ◽  
Positive Control ◽  
Dnase I ◽  
Response Elements ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites ◽  
Dioxin Receptor

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand beta-naphthoflavone. In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.

scholarly journals Ets-2 repressor factor recruits histone deacetylase to silence human cytomegalovirus immediate-early gene expression in non-permissive cells

2005 ◽  
Vol 86 (3) ◽  
pp. 535-544 ◽  
Author(s):  
Edward Wright ◽  
Mark Bain ◽  
Linda Teague ◽  
Jane Murphy ◽  
John Sinclair
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Histone Deacetylase ◽  
Human Cytomegalovirus ◽  
Histone Deacetylation ◽  
Early Gene ◽  
Immediate Early ◽  
Physical Interaction ◽  
Cellular Transcription

Previous work from this laboratory has shown that expression of human cytomegalovirus (HCMV) immediate-early (IE) genes from the major immediate-early promoter (MIEP) is likely to be regulated by chromatin remodelling around the promoter affecting the acetylation state of core histone tails. The HCMV MIEP contains sequences that bind cellular transcription factors responsible for its negative regulation in undifferentiated, non-permissive cells. Ets-2 repressor factor (ERF) is one such factor that binds to such sequences and represses IE gene expression. Although it is not known how cellular transcription factors such as ERF mediate transcriptional repression of the MIEP, it is likely to involve differentiation-specific co-factors. In this study, the mechanism by which ERF represses HCMV IE gene expression was analysed. ERF physically interacts with the histone deacetylase, HDAC1, both in vitro and in vivo and this physical interaction between ERF and HDAC1 mediates repression of the MIEP. This suggests that silencing of viral IE gene expression, associated with histone deacetylation events around the MIEP, is mediated by differentiation-dependent cellular factors such as ERF, which specifically recruit chromatin remodellers to the MIEP in non-permissive cells.

scholarly journals NF-E2 disrupts chromatin structure at human beta-globin locus control region hypersensitive site 2 in vitro.

1996 ◽  
Vol 16 (10) ◽  
pp. 5634-5644 ◽  
Author(s):  
J A Armstrong ◽  
B M Emerson
Keyword(s):  
Control Region ◽  
Chromatin Structure ◽  
Locus Control Region ◽  
Dnase I ◽  
Hematopoietic Cell ◽  
Beta Globin ◽  
Hypersensitive Site ◽  
Dnase I Hypersensitive Sites ◽  
Hypersensitive Sites

The human beta-globin locus control region (LCR) is responsible for forming an active chromatin structure extending over the 100-kb locus, allowing expression of the beta-globin gene family. The LCR consists of four erythroid-cell-specific DNase I hypersensitive sites (HS1 to -4). DNase I hypersensitive sites are thought to represent nucleosome-free regions of DNA which are bound by trans-acting factors. Of the four hypersensitive sites only HS2 acts as a transcriptional enhancer. In this study, we examine the binding of an erythroid protein to its site within HS2 in chromatin in vitro. NF-E2 is a transcriptional activator consisting of two subunits, the hematopoietic cell-specific p45 and the ubiquitous DNA-binding subunit, p18. NF-E2 binds two tandem AP1-like sites in HS2 which form the core of its enhancer activity. In this study, we show that when bound to in vitro-reconstituted chromatin, NF-E2 forms a DNase I hypersensitive site at HS2 similar to the site observed in vivo. Moreover, NF-E2 binding in vitro results in a disruption of nucleosome structure which can be detected 200 bp away. Although NF-E2 can disrupt nucleosomes when added to preformed chromatin, the disruption is more pronounced when NF-E2 is added to DNA prior to chromatin assembly. Interestingly, the hematopoietic cell-specific subunit, p45, is necessary for binding to chromatin but not to naked DNA. Interaction of NF-E2 with its site in chromatin-reconstituted HS2 allows a second erythroid factor, GATA-1, to bind its nearby sites. Lastly, nucleosome disruption by NF-E2 is an ATP-dependent process, suggesting the involvement of energy-dependent nucleosome remodeling factors.

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