Modulation of tk expression in mouse pericentromeric heterochromatin.

Abstract

Constitutive heterochromatin is that part of the chromosome which does not decondense or unravel during interphase (for a review, see reference 2). It is commonly associated with centromeres and contains large amounts of satellite DNA, a class of highly repetitive DNA composed of tandem arrays of simple sequence repeats (for a review, see references 3, 26, and 47). Constitutive heterochromatin is generally associated with transcriptional inactivity. One indication of this is the low density of major gene loci in constitutive heterochromatin (25). Furthermore, genes translocated to the vicinity of heterochromatin are frequently repressed or variegated. In Drosophila melanogaster, this repression has been correlated with the spread of the condensed state from the heterochromatic region to the translocated euchromatin (29), and the probability of repression has been observed to increase with increasing proximity to the heterochromatin. This type of cis effect is classically referred to as position effect or position effect variegation. A similar phenomenon occurs in mice in which autosomal loci translocated to a heterochromatic X chromosome also are observed to be inactivated (6).The molecular mechanism underlying classical position effects remains obscure. A detailed molecular analysis of genes modulated within heterochromatin may provide insights into this intriguing phenomenon. In the long term, such studies may allow the identification of cis-acting elements which play a role in exerting position effects. In this study, we describe a cell line, LC2-3, which can serve as a valuable model system for such studies. LC2-3 is a cell line derived from thymidine kinase-deficient (tk-) mouse L cells (LMTK-cells) satellite DNA was found to immediately flank these tk inserts and, using the tk DNA as a marker, the adjacent satellite DNA was shown to undergo a high frequency of DNA rearrangements (5), a process which is reminiscent of the known fluidity of satellite DNA. In the current study, we characterized three pairs of tk-revertant and tk+ rerevertant derivatives of LC2-3. Our results indicate that one of the tk genes in LC2-3 is probably not expressed but can be reactivated in association with DNA rearrangements and changes in DNA methylation. Through these and other studies in the future, we hope to obtain insights into the molecular mechanism mediating tk repression and, furthermore, to determine whether and how the flanking centromeric heterochromatin may participate in this proc- 5-AzaC treatments. Treatments with 5-azacytidine (5-azaC) were performed by plating 106 cells into a 100-mm dish containing medium supplemented with 5-azaC at a concentration of 0, 1, 3, or 10 ,uM. Cells were exposed to the nucleotide analog for 2 days and subsequently grown for 2 days in medium without 5-azaC. Cells were then harvested from each dish and replated at a density of 5 x 106 cells per 100-mm dish in HAT medium and 200 cells per 100-mm dish in BrdU medium. After 7 to 10 days of growth, colonies were fixed in methanol-acetic ...

Expression of a foreign gene in a line of transgenic mice is modulated by a chromosomal position effect.

“…The same general approach was previously used to study the cause of the inactivation of an HSV TK gene that had become expressionally inert while inserted into the chromosomes of an L-cell line. In that case too, the activity of the gene was restored when it was recovered and reintroduced into the genome of a cell (4). At least two of our secondary transgenics showed some rearrangement of the foreign DNA.…”

“…The HSV TK gene which spawned expressionally inactive variants while resident in L cells was hypermethylated in most cases (4). Similarly, the murine line-specific inactive Mov proviruses studied by Jahner and Jaenisch (21) become hypermethylated during early development and are later selectively demethylated in a line-and tissue-specific manner.…”

Satellite DNA induces unstable expression of the adjacent herpes simplex virus tk gene cotransfected in mouse cells.

“…Cell lines that show phenotypic instability without detectable alterations of the TK sequences have been reported occasionally (10,13,35). Since different cell lines have the transferred genes integrated at different chromosomal sites (7,30,33), the ability of the gene to function in each line would reflect the influence of the surrounding chromosomal sequences.…”

“…The transfected genes are frequently stably expressed and confer on the recipient cells new phenotypic markers; reversion to the original phenotype occurs at low frequency and is commonly the result of loss of the foreign genes. In cell lines that show unstable expression of the exogenous genes, modulation of the transcriptional activity has been related to DNA rearrangements (7,29), changes in the methylation pattern (7-9, 17, 27), or variation of the chromatin structure of the transfected genes (13,35). Since transferred DNA sequences appear to be integrated into the host genome at random sites (6,23,30,33), their expression and regulation may depend upon the new genetic environment into which they are inserted.…”

“…Evidence that the genetic environment can influence gene expression also derives from experiments of DNA-mediated gene transfer (7,29). Genes introduced into mouse fibroblasts by the calcium-phosphate precipitation technique are integrated into the host genome and stably maintained for several generations (19,29,30,33,36,42).…”