Deoxyribonuclease I sensitivity of plasmid genomes in teratocarcinoma-derived stem and differentiated cells.

Murine teratocarcinoma stem cells, unlike most other cell types, do not express major histocompatibility antigens. The steady-state levels of fi-microglobulin and H-2 mRNA from F9-derived teratocarcinoma stem and differentiated cells were examined by blot hybridization using cloned DNA probes specific for these mRNAs. No H-2-or 32-microglobulin-specific RNA was detected in F9 teratocarcinoma stem cells (clone 12-1); thus, F9 teratocarcinoma stem cells (clone 12-1) contain no more than 1/ 10 the H-2 and P2-microglobulin mRNAs of the differentiated daughter cells (clone 12-la). We suggest that this regulation of major histocompatibility antigen expression is due to transcriptional control of the major histocompatibility antigen genes, H-2 and 32-microglobulin. The transcriptional regulation of these genes is accompanied by a change in their DNase I sensitivity. Normally, transcriptionally inactive genes are DNase I resistant, while active genes are DNase I sensitive. In contrast, the silent major histocompatibility antigen genes of teratocarcinoma stem cells are more DNase I sensitive than the active genes of the differentiated cells.The regulation of expression of the major histocompatibility antigens has intrigued immunologists and developmental biologists for many years. These antigens are detected on the surface ofnearly all types ofsomatic cells. Murine teratocarcinoma stem cells, like cells of the early mouse embryo (1-4), do not express these antigens (5-9). However, differentiated cell lines derived from the teratocarcinoma stem cell, like other somatic cells, do express these antigens (5-7). Until recently, the control of the expression of the histocompatibility genes could be analyzed only by serological and biochemical analysis of the histocompatibility protein antigens. With the availability ofcloned molecular DNA probes, we can now examine both the mRNA transcripts and the state of the genomic DNA in teratocarcinoma-derived stem cells and differentiated cells. We report here blot hybridization analysis of RNA extracted from the F9-derived stem and differentiated cell lines, 12-1 and 12-la, respectively, using cloned 832-microglobulin and H-2 probes. In addition, we have examined the DNase I sensitivity of the H-2 and 82-microglobulin genes to determine whether specific murine genes within stem cell chromatin are, like total stem cell chromatin (10), DNase I hypersensitive regardless oftheir transcriptional state. MATERIALS AND METHODSCells. 12-1 teratocarcinoma stem cells were isolated (11) after transfection ofthymidine kinase (Tk)-deficient F9 cells (12) Isolation of Nuclei. All manipulations after collection ofcells were at 4°C. Cells were scraped from glass bottles and washed twice in phosphate-buffered saline and once in reticulocyte standard (RS) buffer (0.01 M Tris-HCl, pH 7.4/0.01 M NaCl/ 3 mM MgCl2) and nuclei were isolated by suspension in RS buffer/0.5% Nonidet P40 (British Drug House, Poole, England) essentially as described by Weintraub and Groudine (21). Nuclei were washed twic...

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“…5B, lane 9). We conclude that H-2 and P2-microglobulin genes, like total chromatin and integrated plasmid genomes (10) …”

Section: Resultsmentioning

confidence: 99%

“…DNase I digestions were carried out by the procedure of Weintraub and Groudine (21) as described (10) [10][11][12][13][14][15] hr, and DNA was isolated from all samples as described (10).…”

Section: Methodsmentioning

confidence: 99%

Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.

Croce¹,

Linnenbach²,

Huebner³

et al. 1981

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

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104

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“…An alternative to a strict gene regulatory network mechanism of lineage priming or pre-patterning, would be a mechanism involving epigenetic regulation. It has been recognised for many years that pluripotent stem cells have a more open chromatin structure than their differentiated derivatives [32] , perhaps permitting ‘leaky’ gene expression. Much more recently it was reported that in ES cells the histones associated with the chromatin of various developmentally regulated genes, notably transcription factors, show bivalent repressive and activation marks [33] , [34] .…”

Section: Discussionmentioning

confidence: 99%

Prepatterning in the Stem Cell Compartment

et al. 2010

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The mechanism by which an apparently uniform population of cells can generate a heterogeneous population of differentiated derivatives is a fundamental aspect of pluripotent and multipotent stem cell behaviour. One possibility is that the environment and the differentiation cues to which the cells are exposed are not uniform. An alternative, but not mutually exclusive possibility is that the observed heterogeneity arises from the stem cells themselves through the existence of different interconvertible substates that pre-exist before the cells commit to differentiate. We have tested this hypothesis in the case of apparently homogeneous pluripotent human embryonal carcinoma (EC) stem cells, which do not follow a uniform pattern of differentiation when exposed to retinoic acid. Instead, they produce differentiated progeny that include both neuronal and non-neural phenotypes. Our results suggest that pluripotent NTERA2 stem cells oscillate between functionally distinct substates that are primed to select distinct lineages when differentiation is induced.

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“…It was reported that chromatin in undifferentiated cells is generally sensitive to DNase I digestion (26). The same workers also studied the DNase I sensitivity of transfectionally introduced genes in teratocarcinoma cells and found that these genes are sensitive to the enzyme (12). I tested the DNase I sensitivity of methylated M-MuLV in EC-Al cells and found that the viral sequence in undifferentiated cells was sensitive to DNase I digestion but became resistant to the enzyme in differentiated cells (K. Iwasaki and 0.…”

Section: Discussionmentioning

confidence: 99%

Suppression of the hypomethylated Moloney leukemia virus genome in undifferentiated teratocarcinoma cells and inefficiency of transformation by a bacterial gene under control of the long terminal repeat.

Niwa¹

1985

Mol. Cell. Biol.

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The Moloney leukemia virus (M-MuLV) genome was introduced into undifferentiated teratocarcinoma cells by transfection of a plasmid with the virus genome linked to pSV2neo, which carries a bacterial drug resistance gene, neo, or by cotransfection with pSV2neo. In the resulting cells, the M-MuLV genome remained hypomethylated, but its expression was suppressed in cells in an undifferentiated state. The pattern of DNA methylation of the viral genome remained unchanged when the cells were induced to differentiate into epithelial tissues. However, spontaneous M-MuLV expression was detected with differentiation of the cells. To determine to what extent the viral long terminal repeat (LTR) was responsible for this suppression in undifferentiated cells, I constructed plasmids in which neo was placed under the control of the promoter sequence of the dihydrofolate reductase gene or the M-MuLV LTR, and compared the biological activities of the plasmids in Ltk-cells and in undifferentiated teratocarcinoma cells. In Ltk-cells, these plasmids were highly efficient in making the cells resistant to selection by G418. However, in undifferentiated teratocarcinoma cells, the M-MuLV LTR promoted neo gene expression at only 10% of the expected efficiency, as compared with the expression of the neo gene under the control of the simian virus to or dihydrofolate reductase promoter. Thus, the mechanisms of gene regulation are not the same in undifferentiated and differentiated teratocarcinoma cells.

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Deoxyribonuclease I sensitivity of plasmid genomes in teratocarcinoma-derived stem and differentiated cells.

Cited by 13 publications

References 29 publications

Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.

Control of expression of histocompatibility antigens (H-2) and beta 2-microglobulin in F9 teratocarcinoma stem cells.

Prepatterning in the Stem Cell Compartment

Suppression of the hypomethylated Moloney leukemia virus genome in undifferentiated teratocarcinoma cells and inefficiency of transformation by a bacterial gene under control of the long terminal repeat.

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