Activation of a silent gene is accompanied by its demethylation

The phenomenon of gene activation by cell fusion makes it possible to study a gene when it passes from a silent to an active state. The relationship between methylation and activation of the mouse albumin gene has been investigated in two types of hybrid clones: mouse lymphoblastoma--rat hepatoma hybrids where activation is very frequent, and mouse L-cell--rat hepatoma hybrids where activation is a rare event. Analysis of the methylation pattern of seven MspI/HpaII sites that occur along the first 8000 bases of the mouse albumin gene has been performed. The entire 5' region is unmethylated only in albumin-producing cells (adult liver and hepatoma); in non-hepatic cells this region is heavily methylated. In hybrids between rat hepatoma cells and mouse cells of mesenchymal origin, the only regular change is the demethylation of the most 5' site (M1), which is systematically observed in clones where expression of the mouse albumin gene has been activated. Demethylation of this site, like activation of the mouse albumin gene, is gene dosage-dependent; it is systematic in the lymphoblastoma--hepatoma hybrids and rare in L-cell--hepatoma hybrids. We conclude that demethylation of this site is tightly coupled with activation of the gene and may well be a necessary prerequisite for activation.     

Extinction of Albumin Gene Expression in a Panel of Human Chromosome 2 Microcell Hybrids

Expression of the serum albumin gene is extinguished in rat hepatoma microcell hybrids that retain mouse chromosome 1. These data define atrans-dominant extinguisher locus,Tse-2,on mouse chromosome 1. To localize the human TSE2 locus, we prepared and characterized rat/human microcell hybrids that contained either human chromosome 1 or chromosome 2, the genetic homologues of mouse chromosome 1. Rat hepatoma microcell hybrids retaining a derivative human chromosome 1 [der 1 t(1;17)(p34.3;q11.2)] expressed their serum albumin genes at levels similar to those of parental hepatoma cells. In contrast, microcell transfer of human chromosome 2 into rat hepatoma recipients produced karyotypically heterogeneous collections of hybrid clones, some of which displayed dramatic albumin extinction phenotypes. For example, albumin mRNA levels in several extinguished microcell hybrids were reduced at least 500-fold, similar to albumin mRNA levels in hepatoma × fibroblast whole-cell hybrids. Expression of several other liver genes, including α1-antitrypsin, aldolase B, alcohol dehydrogenase, and phosphoenolpyruvate carboxykinase, was also affected in some of the microcell hybrids, but expression of these genes was not concordant with expression of albumin. Hybrid segregants were prepared from the albumin-extinguished hybrids, and reexpression of albumin mRNA and protein was observed in sublines that had lost or fragmented human chromosome 2. Finally, expression of mRNAs encoding the liver-enrichedtransactivators HNF-1, HNF-4, HNF-3α, and HNF-3β was not affected in any of the chromosome 2-containing hybrids. These data define and map a genetic locus on human chromosome 2 that extinguishes albumin gene expression intrans,and they suggest that TSE2-mediated extinction is independent of HNF-1, -4, -3α, and -3β expression.     

Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.

Extinction of phosphoenolpyruvate carboxykinase (PCK) gene expression in hepatoma x fibroblast hybrids is mediated by a trans-acting genetic locus designated tissue-specific extinguisher 1 (TSE1). To identify PCK gene sequences required for extinction, hepatoma transfectants expressing PCK-thymidine kinase (TK) chimeric genes were fused with TK- fibroblasts and PCK-TK expression in the resulting hybrids was monitored. Expression of a PCK-TK chimera containing PCK sequences between base pairs -548 and +73 was extinguished in four of five hepatoma transfectants tested, although hybrids derived from one transfectant clone failed to extinguish PCK-TK expression. In contrast, crosses between hepatoma transfectants expressing the herpesvirus TK gene from its own promoter and TK- fibroblasts produced TK+ hybrids; extinction of the transfected TK gene was not observed. Thus, rat PCK gene sequences between base pairs -548 and +73 are sufficient for tissue-specific extinction in hybrid cells. Extinction of PCK-TK gene expression in transfectant microcell hybrids mapped specifically to human chromosome 17, the site of human TSE1.     

Undermethylation at the 5′ end of the albumin gene is necessary but not sufficient for albumin production by rat hepatoma cells in culture

We have measured methylation of the albumin gene in clones of rat hepatoma cells that vary quantitatively in their rates of synthesis of albumin and in variant and hybrid cells that produce no albumin. Although the albumin gene is undermethylated for its entire length in rat liver, only the 5′ end is ever undermethylated in hepatoma cells. Moreover, undermethylation of the 5′ end of the gene appears to be necessary for stable expression of the albumin gene in hepatoma cells. Since undermethylation of this region is found in some variant cells that fail to produce albumin, it is not a sufficient condition for albumin gene expression. Despite the excellent correlation between undermethylation of the 5′ end of the albumin gene and its stable expression, the results argue against the possibility that the methylated state of such genes during development determines whether they will or will not be expressed.     

Expression of differentiated functions in hepatoma cell hybrids. II. Aldolase

A study of aldolases in rat hepatoma clones and subclones has revealed that they synthesize all three forms of aldolase monomers: A (the ubiquitous glycolytic isozyme), B (the form characteristic of the liver) and C, and that in vitro–in vivo passage results in a reversible modulation in aldolase A activity. Three kinds of somatic hybrids, between rat hepatoma cells and either mouse fibroblasts or rat epithelial cells, have been studied. In each case, aldolase B, found only in the hepatoma parent, was absent in the hybrid cells. The absence of aldolase B in the somatic hybrids seems not to be due to trivial factors (species differences, inactivation of all hepatoma aldolase genes, increase in ploidy or loss of chromosomes); it is concluded that extinction of this differentiated function of the hepatoma parent reflects a genetic regulatory phenomenon.     

Somatic cell hybrids between totipotent mouse teratocarcinoma and rat hepatoma cells.

We have produced somatic cell hybrids between totipotent mouse teratocarcinoma cells and rat hepatoma cells. These hybrids were tested for the expression of liver specific functions expressed in the hepatoma cell parent and for their ability to differentiate when injected into nude mice. The results of this study indicate that hybrid cell clones do not resemble either of the parental cells, since they do not produce albumin and tyrosine aminotransferase that are expressed in the rat hepatoma parent, and are incapable of forming either teratocarcinomas or hepatomas when injected in experimental animals.     

Extinction of liver-specific functions in hybrids between differentiated and dedifferentiated rat hepatoma cells.

A cross has been performed between dedifferentiated rat hepatoma cells and the differentiated cells from which they were derived. 10 hybrid clones, containing the complete chromosome sets of both parents, show extinction of 4 liver-specific enzymes: tyrosine aminotransferase (E.C. 2.6.1.5), alanine aminotransferase (E.C. 2.6.1.2), and the liver-specific isozymes of alcohol dehydrogenase (E.C. 1.1.1.1) and aldolase (E.C. 4.1.2.13). Moreover, the 4 hybrid clones examined do not produce albumin . The only function of the differentiated parent which is not extinguished in the hybrid cells is inducibility of the aminotransferases. For 3 of the hybrid clones, extinction of 3 of the 4 enzymes is incomplete, but these clones do not differ in modal chromosome number from those which show more complete extinction of the enzymes. Subcloning of several of the hybrids revealed that the phenotype of the hybrids is very stable; 4 subclones showing reexpression of intermediate levels of the enzymes are characterized. These results show that dedifferentiation of the parental cells is not due to the simple loss of some factor required for the maintenance of expression of differentiated functions, and suggest that dedifferentiation is due to the activation of some control mechanism, whose final effect is negative, and which may be a part of the epigenotype of the embryonic hepatocyte.     

Glucocorticoid responsiveness of hepatoma cell hybrids

The dominance or recessiveness of glucocorticoid responsiveness and albumin production of different hepatoma cell lines were investigated using somatic cell hybrids. The majority of the intraspecific, intraorgan hybrids between glucocorticoid sensitive, tyrosine aminotransferase (TAT) non-inducible, albumin non-secreting parents and glucocorticoid resistant, TAT-inducible, albumin secreting parents were glucocorticoid sensitive, TAT non-inducible and albumin non-producing. The TAT inducibility was extinguished in interspecific, interorgan hybrids of TAT-inducible hepatoma and non-inducible Chinese hamster fibroblast parents. No TAT reexpression was observed among the many independent hybrid clones. The experiments provided evidence for the non-coordinate control of the expression of differentiated functions in hepatoma hybrid cells.     

Activation of production of mouse liver enzymes in rat hepatoma-mouse lymphoid cell hybrids

The production of four liver-specific enzymes (tyrosine aminotransferase or TAT, alanine aminotransferase, aldolase B, and alcohol dehydrogenase) has been analyzed in rat hepatoma-mouse lymphoid cell hybrids containing a 1s or 2s complement of rat chromosomes. The hybrid clones which retain a nearly 2s complement of rat chromosomes show high activity of all four enzymes; those which contain a 1s rat complement show partial or complete extinction of these enzymes. The tyrosine aminotransferase produced by most of the hybrid clones is identifiable as being of both rat and mouse origin, based upon criteria of temperature sensitivity and electrophoretic mobility; antiserum to the rat liver enzyme was used to distinguish the pseudo-TAT produced by the lymphoid parent from liver-TAT produced by hepatoma and hybrid cells. By the criteron of electrophoretic mobility, the liver form (B) of aldolase, produced by only some of the hybrid clones, appears to be composed of both rat and mouse subunits. We conclude that when extinction of tissue-specific proteins does not occur or is only partial in hybrid cells (due to gene dosage effects), the genomes of both parents may be active in directing synthesis of these proteins.     

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.     

A domain of methylation change at the albumin locus in rat hepatoma cell variants.

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.     

Phenotypic and molecular expression of albumin in rat hepatoma X L cell hybrids

A rat hepatoma cell line (H₄A_ZC₂) was characterized with respect to seven liver-specific phenotypes. Ten clones from the fusion of H₄A_ZC₂ and mouse L cell were analyzed for the expression of these phenotypes. The only hepatic function retained by the hybrid clones was rat albumin synthesis which continued at reduced levels relative to the hepatoma parent. Rat albumin cDNA analysis of RNA from parental and hybrid cells indicated that the reduction in albumin production observed in the hybrids was reflected in coordinate reduction of cytoplasmic rat albumin mRNA.     

Tse-2: a trans-dominant extinguisher of albumin gene expression in hepatoma hybrid cells.

Serum albumin gene expression is generally extinguished in hepatoma x fibroblast hybrids. To define the genetic basis of this phenomenon, we screened a panel of hepatoma hybrids retaining different fibroblast chromosomes for albumin production by immunofluorescence. We report that albumin extinction in these clones was strictly correlated with the retention of mouse chromosome 1. Furthermore, albumin was systematically reexpressed in chromosome 1 segregants. These data define a tissue-specific extinguisher locus (Tse-2) that affects albumin gene expression in trans. Two other liver genes, those encoding liver alcohol dehydrogenase and liv-10, were coordinately extinguished with albumin in monochromosomal hybrids that specifically retained mouse chromosome 1.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Analysis of myogenesis by somatic cell hybridization : I. Myogenic competence of homotypic hybrids derived from rat L6 myoblasts

Several authors have described the extinction of myogenic competence in hybrids produced by fusion of myogenic and non-myogenic cells. Interpretations of such experiments rest upon the assumption that extinction does not occur with any appreciable frequency as a non-specific consequence of the cell hybridization process itself. In order to test this assumption we have analyzed the myogenic competence of over 140 independent homotypic hybrid clones produced by PEG-mediated fusion of rat L6 myoblasts. Based upon an evaluation of myotube formation in hybrid colonies, we demonstrate that 99% of primary hybrid clones are myogenic. The fact that 97% of secondary hybrid colonies also differentiate indicates that myogenic competence is a stable characteristic of the hybrids. Four hybrid clones were isolated and expanded for analyses of chromosome numbers, myotube formation, creatine kinase activities, and microfluorimetric DNA determinations of myotube nuclei. Our results demonstrate that polyploid homotypic hybrid cells produced by fusion of non-neoplastic, developmentally determined rat myoblasts retain and express their program of differentiation. This work provides a foundation for future studies which will investigate the expression of myogenic properties in hybrids between myogenic and non-myogenic cells.     

An exogenous albumin promoter can become silent in dedifferentiated hepatoma variants as well as intertypic hybrids

In order to evaluate the ability of an exogenous tissue-specific promoter to undergo the same dynamic changes in activity as the endogenous one, a 400-base pair fragment of the rat albumin proximal promoter, upstream of the bacterial gpt gene, has been introduced into rat hepatoma cells. Four clones containing a single integrated copy of the construct and producing substantial amounts of albumin and of xanthine phosphoribosyltransferase were isolated. These clones were subjected to two treatments known to result in silencing of the albumin gene: selection for dedifferentiated variants, and fusion with L-cell fibroblasts. In most cases, the albumin-negative progeny obtained no longer expressed the gpt gene: the exogenous promoter of 400 base pairs must contain the sequences required to respond to the mechanisms that block activity of the endogenous gene. However, exceptions were observed: the albumin-deficient variants of one clone remained xanthine phosphoribosyltransferase positive, and some of the albumin-negative hybrids from a different clone continued to produce xanthine phosphoribosyltransferase. These cases of dissociation in expression of the endogenous and the exogenous genes indicate that the site of integration of the alb-gpt construct in one clone renders the sequences insensitive to the mechanisms responsible for albumin gene silencing in dedifferentiated variants, and in the other clone to the mechanism of extinction. Consequently, the mechanisms causing gene silencing in variants and in intertypic hybrids must be different.     

Extinction of alpha-fetoprotein gene expression in somatic cell hybrids involves cis-acting DNA elements.

alpha-Fetoprotein (AFP), a liver-specific protein, is extinguished in somatic cell hybrids formed by the fusion of mouse hepatoma cells (BWTG3) with rat fibroblast cells (JF1). Our studies show that the extinction of mouse AFP expression in these somatic cell hybrids may involve at least two cis-acting regulatory domains, i.e., the enhancer elements and a tissue-specific promoter region, which are located in the 5'-flanking region of the AFP gene.     

Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.