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

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.

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Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4028 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4028-4040 ◽

Cited By ~ 234

Author(s):

N Nakajima ◽

M Horikoshi ◽

R G Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Extract ◽

Initiation Site ◽

Dnase I ◽

Hsp 70 ◽

Exonuclease Iii ◽

Nuclear Extracts ◽

Heat Treated ◽

Hypersensitive Sites

Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.452-461.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 452-461 ◽

Cited By ~ 2

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5634 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5634-5644 ◽

Cited By ~ 78

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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Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4028-4040.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4028-4040

Author(s):

N Nakajima ◽

M Horikoshi ◽

R G Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Extract ◽

Initiation Site ◽

Dnase I ◽

Hsp 70 ◽

Exonuclease Iii ◽

Nuclear Extracts ◽

Heat Treated ◽

Hypersensitive Sites

Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.452 ◽

1986 ◽

Vol 6 (2) ◽

pp. 452-461 ◽

Cited By ~ 56

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.

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