scholarly journals Alternative inducers of the rat metallothionein I gene cause distinct changes in chromatin structure in the 5' region of the gene.

1986 ◽  
Vol 6 (7) ◽  
pp. 2576-2581 ◽  
Author(s):  
S J Taplitz ◽  
K L Calame ◽  
H R Herschman
Keyword(s):  
Chromatin Structure ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Dnase I ◽  
Hepatoma Cell Line ◽  
Hypersensitive Site ◽  
Level Control ◽  
Dnase I Hypersensitive Site ◽  
Rat Hepatoma Cell Line ◽  
I Gene

We examined the chromatin structure of the rat metallothionein I gene, both in uninduced cells and in cells induced by heavy metals or dexamethasone, using hypersensitivity to DNase I as an assay. The metallothionein I gene of the H4IIE rat hepatoma cell line, expressed at basal level, has a single DNase I-hypersensitive site. This site maps between putative hormone and basal level control sequences. Induction of the gene by cadmium or zinc resulted in the appearance of a new hypersensitive site near the start site of transcription, in a region near the metal-regulatory elements. In contrast, induction of the metallothionein I gene by dexamethasone did not alter the basal pattern of hypersensitivity. Thus, different mechanisms of induction of metallothionein transcription lead to distinct alterations in the chromatin containing the 5' sequences regulating the expression of this gene.

Alternative inducers of the rat metallothionein I gene cause distinct changes in chromatin structure in the 5' region of the gene

1986 ◽  
Vol 6 (7) ◽  
pp. 2576-2581
Author(s):  
S J Taplitz ◽  
K L Calame ◽  
H R Herschman
Keyword(s):  
Chromatin Structure ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Dnase I ◽  
Hepatoma Cell Line ◽  
Hypersensitive Site ◽  
Level Control ◽  
Dnase I Hypersensitive Site ◽  
Rat Hepatoma Cell Line ◽  
I Gene

We examined the chromatin structure of the rat metallothionein I gene, both in uninduced cells and in cells induced by heavy metals or dexamethasone, using hypersensitivity to DNase I as an assay. The metallothionein I gene of the H4IIE rat hepatoma cell line, expressed at basal level, has a single DNase I-hypersensitive site. This site maps between putative hormone and basal level control sequences. Induction of the gene by cadmium or zinc resulted in the appearance of a new hypersensitive site near the start site of transcription, in a region near the metal-regulatory elements. In contrast, induction of the metallothionein I gene by dexamethasone did not alter the basal pattern of hypersensitivity. Thus, different mechanisms of induction of metallothionein transcription lead to distinct alterations in the chromatin containing the 5' sequences regulating the expression of this gene.

scholarly journals AnCF, the CCAAT Binding Complex ofAspergillus nidulans, Is Essential for the Formation of a DNase I-Hypersensitive Site in the 5′ Region of theamdS Gene

1999 ◽  
Vol 19 (10) ◽  
pp. 6523-6531 ◽  
Author(s):  
Frank M. Narendja ◽  
Meryl A. Davis ◽  
Michael J. Hynes
Keyword(s):  
Chromatin Structure ◽  
Gene Deletion ◽  
Critical Role ◽  
Dnase I ◽  
Open Chromatin ◽  
Hypersensitive Site ◽  
Transcriptional Start Point ◽  
Dnase I Hypersensitive Site ◽  
Ccaat Box ◽  
Ccaat Sequence

ABSTRACT The CCAAT sequence in the amdS promoter ofAspergillus nidulans is recognized by AnCF, a complex consisting of the three evolutionary conserved subunits HapB, HapC, and HapE. In this study we have investigated the effect of AnCF on the chromatin structure of the amdS gene. The AnCF complex and the CCAAT sequence were found to be necessary for the formation of a nucleosome-free, DNase I-hypersensitive region in the 5′ region of theamdS gene. Deletion of the hapE gene results in loss of the DNase I-hypersensitive site, and the positioning of nucleosomes over the transcriptional start point is lost. Likewise, a point mutation in the CCAAT motif, as well as a 530-bp deletion which removes the CCAAT box, results in the loss of the DNase I-hypersensitive region. The DNase I-hypersensitive region and the nucleosome positioning can be restored by insertion of a 35-bp oligonucleotide carrying the CCAAT motif. A DNase I-hypersensitive region has been found in the CCAAT-containing fmdS gene and was also hapE dependent. These data indicate a critical role for the AnCF complex in establishing an open chromatin structure in A. nidulans.

Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation

1987 ◽  
Vol 7 (11) ◽  
pp. 3863-3870
Author(s):  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Hepatoma Cell Line ◽  
Butyrate Treatment ◽  
Dnase I Sensitivity ◽  
Induced Changes ◽  
I Gene ◽  
Nuclease Sensitivity

We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

scholarly journals Butyrate-induced changes in nuclease sensitivity of chromatin cannot be correlated with transcriptional activation.

1987 ◽  
Vol 7 (11) ◽  
pp. 3863-3870 ◽  
Author(s):  
B W Birren ◽  
S J Taplitz ◽  
H R Herschman
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Dnase I ◽  
Micrococcal Nuclease ◽  
Hepatoma Cell Line ◽  
Butyrate Treatment ◽  
Dnase I Sensitivity ◽  
Induced Changes ◽  
I Gene ◽  
Nuclease Sensitivity

We examined in the H4IIE rat hepatoma cell line the relationship between butyrate-induced changes in the nuclease sensitivity of chromatin and changes in transcriptional activity of specific genes. The butyrate-inducible metallothionein I (MT-I) gene underwent a dramatic increase in DNase I sensitivity after 3 h of butyrate treatment. However, genes not transcribed in H4IIE cells underwent the same changes in DNase I sensitivity. Thus, butyrate-induced increases in DNase I sensitivity are not sufficient for the transcriptional activation of a gene. Butyrate treatment has also been reported to alter the sensitivity of sequences to micrococcal nuclease (MNase) in a manner reflecting their tissue-specific expression. Butyrate exposure caused increased digestion of the MT-I gene by MNase. However, butyrate-induced MNase sensitivity also occurred for genes which are neither transcribed in untreated cells nor butyrate inducible. Moreover, cadmium, a potent transcriptional activator of the MT-I gene, does not alter the sensitivity of the MT-I gene to MNase. Thus, the butyrate-induced alterations in MNase sensitivity are neither sufficient for, necessary for, nor indicative of transcriptional activation.

Activation of the silent endogenous cholesterol-7-alpha-hydroxylase gene in rat hepatoma cells: a new complementation group having resistance to 25-hydroxycholesterol

1991 ◽  
Vol 11 (4) ◽  
pp. 2049-2056
Author(s):  
J K Leighton ◽  
S Dueland ◽  
M S Straka ◽  
J Trawick ◽  
R A Davis
Keyword(s):  
Cell Line ◽  
Cholesterol Biosynthesis ◽  
Hepatoma Cell ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Complementation Group ◽  
Hepatoma Cell Line ◽  
Cytotoxic Action ◽  
Hydroxylase Activity ◽  
Rat Hepatoma

The oxysterol 25-hydroxycholesterol acts both as a regulatory sterol determining the expression of genes governed by sterol regulatory elements and as a substrate for 7-alpha-hydroxylase, the first and rate-limiting enzyme in the bile acid synthetic pathway. Most wild-type nonhepatic cells are killed by the cytotoxic action of 25-hydroxycholesterol. In contrast, liver cells, which express 7-alpha-hydroxylase activity, are resistant to killing by 25-hydroxycholesterol. We examined the possibility that selection for resistance to 25-hydroxycholesterol might lead to the derivation of a cell line expressing 7-alpha-hydroxylase. A rat hepatoma cell line (7-alpha-hydroxylase minus) was transfected with human DNA and screened for resistance to 25-hydroxycholesterol. Although parental hepatoma cells were all killed within a week, a 25-hydroxycholesterol-resistant cell line (L35 cells) which showed stable expression of 7-alpha-hydroxylase activity and mRNA was obtained. These cells exhibited normal inhibition of cholesterol biosynthesis by 25-hydroxycholesterol. Blocking 7-alpha-hydroxylase activity with ketoconazole also blocked the resistance of L35 cells to 25-hydroxycholesterol. Isolation of microsomes from these cells showed levels of 7-alpha-hydroxylase activity (22.9 pmol/min/mg of protein) that were comparable to the activity (33.2 pmol/min/mg) of microsomes isolated from the livers of rats killed during the high point of the diurnal cycle. Parental cells had no detectable activity. These data show a new complementation group for 25-hydroxycholesterol resistance: expression of 7-alpha-hydroxylase. Dexamethasone increased both the activity and the cellular content of mRNA coding for 7-alpha-hydroxylase. Since dactinomycin blocked the ability of dexamethasone to induce mRNA, active transcription is required. Southern analysis of genomic DNA showed that L35 cells contain the rat (endogenous) gene but not the human gene. Furthermore, the RNA expressed by L35 cells is similar in size to rat RNA and is distinct from the human form of 7-alpha-hydroxylase. The combined data indicate that L35 cells are resistant to 25-hydroxycholesterol because they express 7-alpha-hydroxylase. The mechanism responsible involves activation of the endogenous (silent) gene of the parental rat hepatoma cell.

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