In vitro mouse mammary tumor virus transcription from chromatin A system to study the mechanism of action of glucocorticoid hormones

Crépin, Michel

doi:10.1016/0014-5793(77)80703-5

Cited by 6 publications

(6 citation statements)

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“…When chromatin from these treated cells is transcribed with calf thymus RNA polymerase B, viral RNA synthesis in vitro is increased up to 10-fold [21]. We have also observed with chromatin from cells treated for 6 h a stimulatory effect of bound non-histone proteins (Table 1 ; experiment no.…”

Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Moussupporting

confidence: 62%

“…Because the concentration of mouse mammary tumor virus RNA can be determined by hybridization with a complementary cDNA [14], it is possible to measure the transcription of viral RNA in vitvo by E. coli RNA polymerase and eukaryotic RNA polymerase B. Whereas the prokaryotic enzyme transcribed viral RNA sequences very poorly, the eukaryotic RNA polymerase B synthesizes a significant amount of viral RNA [21]. In this system, nonhistone proteins bound to the chromatin failed to affect the transcription by the E. coli enzyme.…”

Section: Discussionmentioning

confidence: 99%

“…The concentration and the time necessary for annealing of cDNA are described in Materials and Methods. It has been previously shown by us [21] and others [22] that the amount of viral RNA in nuclei can be estimated with a 10% error. From hybridization curves (Fig.…”

Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Mousmentioning

confidence: 99%

“…Previous studies have shown that viral RNA can be synthesized in vitro using chromatin [21] or nuclei [22] from G R mammary cells and RNA polymerase B. In contrast to E. coli RNA polymerase, an excess of exogenous RNA polymerase B purified from calf thymus synthesizes viral RNA very efficiently.…”

Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Mousmentioning

confidence: 99%

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Regulation of Transcription by DNA‐Bound Non‐histone Nuclear Proteins

Crépin¹,

Dastugue²

1979

European Journal of Biochemistry

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Purified non-histone proteins from mouse mammary cells bind specifically to homologous DNA or chromatin. Complexes of non-histone protein with DNA or chromatin, isolated on agarose columns, were transcribed with both Escherichia coli RNA polymerase and RNA polymerase B from calf thymus. The fact that complexing of DNA with non-histone proteins increases transcription by E. coli RNA polymerase but not by RNA polymerase B suggests different mechanisms of transcription by these two enzymes. Similar experiments with mouse and Drosophila chromatin indicate that non-histone proteins specifically stimulate the transcription of mouse chromatin by RNA polymerase B. Non-histone proteins stimulate the transcription of mouse mammary tumor virus sequences in chromatin by RNA polymerase B but not by E. coli RNA polymerase. We conclude that those non-histone proteins bound specifically to chromatin are able to activate the transcription of specific genes by eukaryotic RNA polymerase.The non-histone proteins of chromatin are believed to include molecules capable of regulating gene activity. This view was first suggested by the fact that the synthesis of nuclear non-histone proteins increases during experimental gene activation [l]. A fraction of the heterogeneous non-histone proteins is highly tissue-specific [2], and interacts preferentially with homologous DNA [3]. Teng et al. [4] and Shea and Kleinsmith [5] described a protein fraction from rat liver nuclei that stimulates transcription of DNA in vitro with Escherichia coli RNA polymerase. A similar fraction isolated from Ehrlich ascites tumor cells binds selectively to homologous DNA and enhances transcription in an eukaryotic RNA polymerase B system [6,7].While experiments with reconstituted chromatin transcribed in vitro emphasize a role of these nonhistone proteins in the transcription of tissue-specific RNA species [S], no direct evidence exists that these effects depend upon interaction of the protein with DNA. Moreover, some transcription experiments in vitro, using eukaryotic DNA and E. coli RNA polymerase, show a very poor degree of fidelity compared to a cell-free system using chromatin and eukaryotic RNA polymerase B. Although the E. coli polymerase is able to distinguish sequences of chromatin tranEnzymes. DNAse I or deoxyribonucleate 5'-oligonucleotidylhydrolase (EC 3.1.4.5); DNA-dependcnt RNA polymerase (EC 2.7.7.6); single-strand-specific nuclease S1 (EC 3.1.4.21). scribed in vivo [9], it binds to different sites in chromatin compared to eukaryotic RNA polymerase [lo] and often transcribes the incorrect strand [ll].In order to analyze further the role of the nonhistone proteins in regulating transcription, we have isolated specific complexes formed by DNA or chromatin with non-histone proteins (see the preceding paper [12]). The transcription of homologous and heterologous complexes with E. coli RNA polymerase and eukaryotic RNA polymerase B purified from calf thymus has been compared. To study the effect of non-histone proteins on the active genome, we ha...

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Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Moussupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Mousmentioning

confidence: 99%

Section: Effect Of Bond Non-histone Proteins On the Synthesis Of Mousmentioning

confidence: 99%

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Regulation of Transcription by DNA‐Bound Non‐histone Nuclear Proteins

Crépin¹,

Dastugue²

1979

European Journal of Biochemistry

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“…Glucocorticoids also regulate the transcription of integrated murine mammary tumor virus (MMTV) proviral DNA in various cell types (5,6) including lymphoid cell lines (7). The availability of lymphoid cell variants having different sensitivities to the lytic effect ofglucocorticoids allows the study of MMTV expression as a function of glucocorticoid sensitivity.…”

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confidence: 99%

Demethylation and expression of murine mammary tumor proviruses in mouse thymoma cell lines.

Mermod¹,

Bourgeois²,

Defer³

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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Murine mammary tumor virus (MMTV) expression is analyzed in a T-lymphoid cell line (TIMI) sensitive to the killing effect of glucocorticoids and in two of its variants, one resistant (TIMir) and one supersensitive (T MI"5) to glucocorticoidinduced lymphocytolysis. In the TIM, line, MMTV is expressed and induced approximately 10-fold by short treatment with dexamethasone. Southern blot analyses of restriction enzyme digests of DNA Glucocorticoids induce in a-variety of tissues specific responses by mechanisms that appear to be similar to those ofother steroid hormones. Glucocorticoids induce cytolysis ofsome murine cell lines derived from malignant lymphoid T cells (1, 2). This response is mediated by the glucocorticoid receptor, which interacts with chromatin, but the mechanism ofcell lysis remains unknown. Lymphoid cells allow a genetic approach to study of the mechanism of glucocorticoid hormone action because variants resistant to killing can be selected easily. A number of resistant variants have been isolated and characterized from S49 and W7 cell lines (1-3). More recently, variants supersensitive to glucocorticoid-induced killing have been isolated from W7 and TIM, cell lines (4).Glucocorticoids also regulate the transcription of integrated murine mammary tumor virus (MMTV) proviral DNA in various cell types (5, 6) including lymphoid cell lines (7). The availability of lymphoid cell variants having different sensitivities to the lytic effect ofglucocorticoids allows the study of MMTV expression as a function of glucocorticoid sensitivity. Therefore, we have analyzed the control of MMTV RNA concentration by dexamethasone in wild-type, resistant, and supersensitive TIM, cell lines by using cloned MMTV sequences.There is evidence that modification of DNA such as methylation may be correlated with changes in gene expression (8), in particular, in MMTV proviral DNA of mouse tissue (9). By using methylation-sensitive restriction enzymes to detect specific methylation of.MMTV sequences, we have compared the methylation of sites within or flanking the long terminal repeat (LTR) (10) with that of a site within the MMMTV envelope gene.In this paper, we describe the regulation of MMTV RNA synthesis and the methylation ofthe MMTV LTR.and envelope sequences in sensitive, supersensitive, and resistant thymoma cell lines. We present evidence that the expression of endogenous proviruses is different in the various cell lines and appears to correlate with methylation of the LTR. t To whom reprint requests should be addressed. MATERIALS AND METHODS Cell 110The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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