Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism

Cell, Vol. 39, 459-467,

December

1984 (Part 2), CopyrIght

0 1984 by MIT

0092-8674/84/i

30459-09 $02.00/O

Genes with Promoters in Retrovirus Vectors Can Be Independently Suppressed by an Epigenetic Mechanism Michael Emerman and Howard M. Temin McArdle Laboratory for Cancer Research University of Wisconsin-Madison Madison, Wisconsin 53706

Summary We have constructed retrovirus vectors containing two genes, each with its own promoter, whose products can be selected in vivo. In cell clones harboring a single copy of this bicistronic provirus, we find that the 3’ gene is usually suppressed when there is selection for expression of the 5’ gene, and the 5’ gene is usually suppressed when there is selection for expression of the 3’ gene. We find that the suppression is epigenetic, reversible, and cis-acting, and is influenced by the strength or position of the different promoters. This type of gene suppression is novel for its action on a gene that can be either upstream or downstream to an adjacent active gene. Introduction Retroviruses are RNA viruses that replicate through a DNA intermediate. One step in the usual retrovirus life cycle is integration into the cell genome to form a provirus. Once integrated, the provirus is maintained as a stable cellular genetic element. This property of retroviruses makes them attractive vectors for the insertion of exogenous genes into the chromosomes of cells. Because an integrated retrovirus provirus is subject to cellular processes that affect the expression of chromosomal genes, retrovirus vectors can be used in the analysis of these cellular processes. For example, we have adapted the genome of spleen necrosis virus, an avian retrovirus, for use as a vector by inserting exogenous DNA in place of some viral sequences (Shimotohno and Temin, 1981). We have been interested in determining what effects adjacent cistrons have on each other. In a previous paper (Emerman and Temin, 1984) we found that proviruses in cell clones that had been established by infection with a retrovirus containing three promoters and two genes always had deletions that included one of the promoters. Deletions were never found, on the other hand, in proviruses from cell clones established by infection with a retrovirus that contained two promoters and two genes. In the present study we looked for effects of expression of one cistron on the expression of a nearby cistron in integrated retrovirus vectors that contain two promoters and two selectable genes. We have derived several retroviruses with both the neomycin-resistance gene (neo, which codes for resistance in eucaryotes to the drug G418; Colbere-Garapin et al., 1981; Southern and Berg, 1982) and the herpes sim-

plex virus thymidine kinase gene (t/o. Each gene is transcribed from a separate promoter in our viruses (Figure 1). We find that when a single provirus is integrated into the cell genome and we select with the appropriate drug for cells that require expression of the 5’ gene, most cells in a cell clone are killed when selective medium is changed so that expression of only the 3’ gene is required. Likewise, when we select for cells that require expression of the 3’ gene, and then change the selective medium so that expression of only the 5’ gene is required, most cells in a cell clone are killed. We suggest that when we select for expression of one gene in the integrated provirus the other gene is usually entirely or partially inactivated relative to the amount of expression necessary for growth in selective medium (defined as suppression). We show that this suppression is &acting, and by recovering virus from infected cells, we show that the suppressions is epigenetic. This phenomenon of independent “upstream” and “downstream” suppression represents a novel form of gene control. Results Retroviruses with Two Selectable Genes and Two Promoters Have Genetic Transforming Activity for Both Genes We constructed several pBR322-based plasmids that contain within a deleted retrovirus genome two genes selectable in eucaryotic cells (Figure 1). Plasmid pMEl11 has the neo gene from the bacterial transposon Tn5 (Jorgensen et al., 1979) placed 5’ to the herpes simplex virus thymidine kinase (tk) gene. Because the neo gene does not have a eucaryotic promoter, it is transcribed from the promoter in the spleen necrosis virus long terminal repeat (LTR). The tk gene, on the other hand, has been inserted into the retroviral genome with its own promoter. The signals in the tk gene for poly(A) addition have been removed (Shimohtohno and Temin, 1981). In the plasmids pME123 and pME124 the tk gene without its promoter is in the same position as the neo gene in pMEll1. The neo gene is 3’ to the tk gene and adjacent to the tk promoter. The plasmid pME124 differs from pME123 in that the tk promoter-neo gene is inverted relative to its position in pME123. Thus, in pME124, transcription from the tk promoter into the neo gene is in the opposite direction from transcription that initiates in the LTR. Infectious virus was recovered from these plasmid constructs by cotransfection of chicken embryo fibroblasts with the respective plasmid and cloned wild-type REV-A to provide necessary transacting factors. The resultant virus stocks were used to infect BRL TK- cells to determine the TK transforming titer of the virus, to infect D17 cells (or BRL TK- cells, see Experimental Procedures) to determine the NE0 transforming titer of the virus, and to infect chicken embryo fibroblasts to measure the amount of virus

Cell 460

TITER OF RECOVEREDVIRUS

ME III

6x IO4

2 x 106

ME 121

I x 106

0

ME123

4 x 105

8~10~

Table 1. Genetic Transformation

of BRL Cells by ME1 11 Virus

Selection

Number of Colonies/ml ME1 11 Virus Stock

G418

1.5 x 105

HAT

2x

IO4

G418 and HAT

3x

IO2

Selections dures.

and infections

were done as described

of

in Experimental

Proce-

ME III VIRUS 5 x 105

ME 124

SELECT TK+ CELLS

Figure 1, Restriction Recombinants

Enzyme Cleavage

Site Maps of Spleen Necrosis

INFECT

+ BRLTK-

7x10+

NEOS CELLS

g

\

SELECT NEOR CELLS

Virus

Open boxes represent the viral LTRs, lines represent SNV sequences, black-dotted boxes represent the herpes simplex virus thymidine kinase gene transcriptional promoter, white-dotted boxes represent the tk coding sequences, and slashed boxes represent the Tn5 neomycin-resistance gene. All plasmids were constructed in pBR322 (not shown in this figure). Virus titers were determined as described in Experimental Procedures. TK TU stands for thymidine kinase transforming units and is defined as the number of TK- cells converted by infection to a TK+ phenotype. NE0 TU stands for neomycin transforming units and is defined as the number of G41 a-sensitive cells converted by infection to G418 resistance. The titers were corrected for the relative virus production as described in Experimental Procedures. The relative extent of virus production was: SW272, 1.0; ME1 11, 0.3; ME121, 1.3; ME123, 0.7; ME124, 1 .O. The direction of transcription is from left to right in all cases except in ME1 24, where transcription of neo is from right to left as indicated by the arrow.

production and the structure of the recovered viruses All of the viruses with both genes displayed both TK and NE0 transforming activities (Figure 1). Furthermore, the amount of activity was correlated with the promoter that was 5’ to the coding sequences of the gene (that is, the proviral LTR or the tk promoter). For example, the TK transforming titer is approximately lo-fold higher when the tk gene is transcribed by the LTR (ME121, MEI23, and ME124) than when it is adjacent to its own promoter (ME1 11). Likewise, the NE0 transforming activity of ME1 11 is more than 20fold higher than that of ME1 23 and ME1 24. Expression of the neo gene in ME124 shows that transcription occurs from the internal tk promoter. In a previous paper we found that strong promoters in the opposite transcriptional orientation to the LTR reduced the virus titer (Bandyopadhyay and Temin, 1984a). Presumably, this was not the case for ME124 because the tk promoter is a weak promoter in chicken cells. Cell Clones Infected with a Retrovirus That Has Two Selectable Genes and Two Promoters Usually Do Not Fully Express Both Genes The ME11 1 virus stock transforms TK- cells to a TK+ phenotype and NEOS cells to a NEOR phenotype. However, we observed that when we infected cells and then selected in both HAT (for TK+) and G418 (for NEOR) there was greater than a 50-fold reduction in the number of surviving colonies than if we selected in just G418 or HAT alone

PICK ISOLATED COLONIES ‘L

GROW UNDER TK+ f SELECTION

0.5- Ix IO5 CELLS

SE+;$T CELLS

a

0.5- Ix IO5 CELLS

SELE$jT NO NE0 SELECTION CELLS

b

c COUNT

GROW UNDER NEOR SELECTION

SELECT SELECT NO TK+ NEOR SELECTION CELLS CELLS

d COLONI

e

f

ES

Figure 2. Protocol for Establishing Cell Clones under One Condition of Sefection and Testing the Cells for Abilrty to Grow under the Second Condition of Selection See Experimental Procedures for details. Cells were infected at a multiplicity of infection of about 0.0001 transforming units per cell. When cell clones had grown to 0.5-I .O x 1O5 cells under the first condition of selection, they were typsinized, diluted, and replated at about 104, 103, and IO* cells per plate under the three types of selection, The letters a-f represent numbers of colonies and will be referred to in the legend to Table 2.

(Table 1). Moreover, many of the colonies that did survive this double selection grew poorly. Therefore, we wished to determine whether individual cell clones with only one copy of each gene in each cell (that is, one provirus) could display both TK+ and NEOR phenotypes. Our protocol for this determination is outlined in Figure 2. The BRL cells were infected at a low multiplicity with ME1 11 virus. Selective media was added such that on some plates the TK+ cells would survive, and on other plates the NEOR cells would survive. Well-isolated colonies were picked from the plates and grown to about 0.5-1.0 x lo5 cells in the presence of selection. The clones were then diluted and replated under three selection conditions: for the initially selected gene, for the second gene, or with no selection. The number of colonies’ that grew after replating under each of the three conditions was then counted. These results (see below) are presented in Table 2 as the number of colonies that grew when the cells were replated in selection divided by the number of colonies that grew when the cells were replated in no selection. Table 2 (top) shows that ME1 11 -infected cell clones that



Gene Suppression in Retrovirus Vectors 461

had initially been selected for the NEO R phenotype usually could not grow when subsequently placed under selection for the expression of the tk gene. For example, when cell clones were picked from a population of BRL cells that had been infected with ME111 and selected for G418 resistance, seven of the 12 clones would not grow when replated under selective conditions that required expression of the tk gene . When the other five of the 12 cell clones that had been picked initially for neo expression were placed under selective conditions for tk expression, they had only 1%-9% of the plating efficiency in HAT (that is, conditions that required the TK+ phenotype) compared to nonselective conditions . In the same manner, we also tested cell clones initially selected for expression of the TK+ phenotype for subsequent expression of the NEO R phenotype (Table 2) . We found suppression of the NEO R phenotype in 10 of the 12 clones picked for tk expression . These 10 clones had only a 2%-50% relative plating efficiency when subsequently tested for expression of the NEO R phenotype . Two of the cell clones, however, were able to grow in selective conditions for the second gene nearly as well as they grew under nonselective conditions . These two also formed as many colonies when placed under both selective conditions simultaneously as in nonselective conditions (data not shown) . For all cell clones including these two, the colonies formed in the second selection were smaller, and

Table 2 . Ability of Cell Clones Genetically Transformed by ME 111

and

ME123 Virus to Grow under Selection for the Second Gene Ratio of cells that grow in second 1st Selection

2nd Selection

selection :cells that grow in no selection in 24 independent clones (x 100)

ME111 = LTR-neo gene ; tk promoter-tk gene

neo

tk

9, 9, 4, 2, 1, <0 .01, <0 .01, <0.01, <0.01, <0 .01, <0 .01, <0.01' (average value = 2% ±1%)

tk

neo

sometimes of a different morphology than those replated in no selection or in the first selective medium . Thus in 22 out of 24 cell clones initially selected for a phenotype that is determined by expression of one cistron on the bicistronic ME 111 provirus, the subsequent expression of the second phenotype (and therefore the second cistron) is suppressed . We also infected BRL cells, selected for expression of one gene, and when colonies had grown to about 50 cells, replaced the medium with medium that would require expression of the second gene for the colonies to survive . Colonies were marked on the tissue culture plate and observed with a microscope . We found that 75% of the colonies (15 of 20) died, and the ones that did not die grew more slowly than colonies on a control plate that was not placed in the second selective medium (data not shown) . This finding, and those shown in Table 1, indicate that the suppression of the second gene must occur early after infection . Subpopulations of Cells Exist within the Cell Clones with Phenotypes That Differ from That of the Whole Population Cell clones were picked from well isolated colonies and were shown to have a single provirus (see Figure 4A) . However, by the time single cells had grown to 1 X 10 5 cells, subpopulations existed that differed from the population as a whole . Figure 3 shows two examples of different subpopulations in the cell clones . In example I, an ME111infected TK+ cell clone was found to form only 10% of the number of colonies in media with G418 as in media with HAT (or media without drug) . However, when one of the NEOR colonies was picked, grown to a small size in G418, and then retested, it was found to form just as many colonies when diluted and replated under selective conditions for neo as for tk. This example shows that a subpopulation of cells existed in the cell clone that had both genes active . This phenomenon was observed in six out of ten ME1 11 -infected cell clones tested (data not shown) .

100, 95, 50, 35, 30, 20, 15, 10, 9, 9, 3, 2b (average value = 31% ± 9%) I

II

ME123 = LTR-tk gene ; RAT (ME III) TK+ CLONE

tk promoter-neo gene

neo

tk

100, 40, 20, 4, 1, 0 .1 (average value = 28%

tk

neo

10, 10, 10, 4, <0 .01, <0 .01 (average value

-h 13%)

= 6% ± 2%) Experimental protocol is diagrammed in Figure 2 . Data are presented for individual clones .

'

RAT (ME III) NEO R

. SELECT NEO N

10% NEO N

9 % TK+

i ISOLATE RAT (ME III) NEO

i

N CLONE

ISOLATE RAT (MEIII) TK+ CLONE

-'SELECT TK+

a

The ratio of d:f in Figure 2, that is, the ratio of the number of cells that grew in tk on replating a clone selected for growth in neo: the number of cells that grew without drug selection on replating the same clone multiplied by 100 . The ratio of e:f in Figure 2 was 70% -L 6% with a range of 120% to 60% for all clones . I The ratio of b:c in Figure 2, that is, the ratio of the number of cells that grew in neo on replating a clone that was selected for growth in tk: the number of cells that grew without drug selection on replating the same clone multiplied by 100 . The ratio of a:c in Figure 2 was 100% ± 10% with a range of 130% to 60% for all clones .

CLONE

``SELECT TK+

100% TK+

`SSELECT NEO N 10°/n

NEO N

Figure 3 . Examples of the Behavior of Subpopulations in ME111 VirusInfected Cell Clones The cell clones are the same as those shown in the top section of Table 2 . Cells were grown to 0 .5-1 .0 x 105 cells in the presence of HAT or G418 before being tested for their ability to grow in the second selection . Similarly, subclones with the ability to grow in the second selection were grown to 0.5-1 .0 x 10 5 cells in the presence of HAT or G418 before being tested for retention of the ability to grow in the first selection .

Cell 462

In example II we found that a subpopulation could be grown that had reversed the inhibition of the second gene and now was inhibited for growth under selective conditions for the first gene. In this case an ME1 11 -infected NEOR cell clone was grown to 1 x lo5 cells and found to form only 9% of the number of colonies in HAT as under nonselective conditions. However, when one of the TK+ colonies was picked, grown to 1 x IO5 cells in HAT, and retested for plating efficiencies in G418, HAT, and no selection, it was found that now the population grew as well in HAT as in no selection, but only grew one-tenth as well in G418 as in no selection. Thus the suppression had been switched from the tk gene to the neo gene. This phenomenon was observed in two out of ten cell clones tested (data not shown). We wished to determine whether the suppression was stable in the absence of selection, For this reason we took 12 of the cell clones shown in Table 2, diluted them to IO100 cells, grew them to 106-lo7 cells in the absence of selection, and then retested them for expression of NEOR or TK’ phenotypes. We found that in all clones the suppression was stable in the absence of selection. That is, cell clones initially selected for NEO* phenotype were still NEOR, but were usually not TK+, and cell clones initially selected for TK+ phenotype were still TK+, but were usually not NEOR (data not shown). This result indicates that the suppression is a stable phenomenon. Nonetheless, as indicated in Figure 3, variants can arise that have circumvented the suppression.

A

B

abcdef

a

bcdef

ORIGINkbp 23 I9.4 6.6 -

23 I 9.46.6 -

2.8 -1.9

MEIII Hind sac

III I

_____

I -2.6

kbpFl.Skbp+

probe

r

I

Figure 4. Southern Blot of Genomic DNA from BRL(ME111) Hybridized with a tk-Specific Fragment

Cell Clones

Lanes a-f are cell clones shown at the top of Table 2. Lanes a-d were selected for NEOs phenotype. The percentages of cells that could grow in HAT were: a, 4%; b, 1%; c and d,
virus do not account selectable gene.

for the suppression

of the second

The Proviruses in Clones of Cells Infected with ME1 11 Show no Gross Rearrangements

The Suppression of the Second Selectable Gene Is an Epigenetic and cis-Acting Phenomenon

Because our previous work showed that selection for expression of a-gene in a retrovirus vector can lead to viruses with deletions in their genome (Emerman and Temin, 1984) we characterized the ME1 11 proviruses in our cell clones to determine their gross structure. First we determined the number of proviruses in the cell clones. Figure 4A shows a Southern blot of DNA from some representative individual cell clones, digested with Hind Ill and hybridized with tk-specific DNA. The recognition site for Hind III is in the viral genome 5’ to the tk gene. Thus each band in Figure 4A represents a different provirus because the 3’ Hind Ill site is at a unique site in cellular DNA. Figure 4A shows that each clone has only one provirus. Therefore, the phenomenon of suppression of the second selectable gene in these ME1 11 -infected ceil clones occurs at a single genetic locus. Figure 48 shows a Southern blot of Sac l-digested cellular DNA from ME1 11 -infected cell clones that has been hybridized with tk-specific DNA. Unrearranged proviruses should give two internal bands with a tk probe. Every cell clone tested, even those in which there was no detectable tk activity (assayed by the ability of cells to grow in HAT medium) showed internal Sac I bands of the expected size. This result indicates that large deletions (>200 bp) or other rearrangements in the integrated pro-

The suppression of the second selectable gene (that is, tk for cell clones initially selected for neo, and neo for cell clones initially selected for tk) is either the result of a genetic event-for example, small mutations that would inactivate the second gene-or the result of an epigenetic event. To differentiate between these possibilities, we recovered virus from the ME1 11 -infected cell clones and determined the biological activity of the recovered virus. If the suppression of the second selectable gene is the result of a genetic event at the proviral locus, then all the virus recovered from cells of a particular cell clone would also bear this mutation, and the recovered virus would have a decreased ability to transform cells because of its mutant gene. Specifically, the ratio of TK transforming units to NE0 transforming units would be different than it would be for the parental virus stock. (For example, if cells could only grow 1% as well in HAT as in G418, then one might expect the ratio TK:NEO transforming units of virus recovered from these cells to be 1% of wild-type ME1 11). If, on the other hand, the suppression of the second selectable gene is an epigenetic process, then recovering virus from the ceils would regenerate a virus stock with the same ratio of TK transforming units to NE0 transforming units as the parental virus stock. BRL cells and other rat ceils are semipermissive for

Gene Suppression in Retrovirus Vectors 463

growth of spleen necrosis virus . They can be infected, but they do not shed significant amounts of virus (Keshet and Temin, 1979 ; M . Emerman, personal observations) . The scheme to recover virus from these cells, outlined in Figure 5, involved fusing the BRL cell clones to chicken embryo fibroblasts chronically infected with REV-A to stimulate virus production . This procedure allowed recovery of a low-titer virus stock . To obtain a stock of virus with a high titer of TK and NEO transforming units, we infected D17 cells, a dog cell line permissive for virus growth, and selected G418-resistant cell clones that produced high titer virus stocks . The virus recovered from the cell clones was titered on BRL TK - cells for TK transforming activity and on D17 cells for NEO transforming activity (Table 3) . Virus recovered from every cell clone, even those NEO R clones in which no cells would grow in HAT, had the same genetic transforming activity as the parental virus stock . In the NEOR clones the LTR is always active because it must be used to transcribe the neo gene . Because there is no selective pressure for expression of the tk gene during the fusion or subsequent virus growth, the fact that the recovered virus has the same TK transforming activity as the parental virus stock indicates that the source of suppression of the 3' (tk) gene must be epigenetic, rather than genetic . Virus was rescued from the TK+ clones at about the same efficiency as from NEO R clones and was also wildtype (data not shown) . Therefore, the LTR could not have been genetically inactivated . To determine whether the suppression worked in cis or in trans, the following experiment was done . We infected the parental BRL cells (TK -,NEOs) and cells from two BRL cell clones that contained an ME 111 provirus, but were phenotypically TK - ,NEOR (cell clones from Table 2 in which <0 .01 % of the cells grew in HAT) with various dilutions of the ME111 virus stock . Cells were then placed under HAT selection and the number of TK+ colonies was counted 1 week later. If the suppression is a trans effect, then the BRL(ME1 11) TK - ,NEOR cells would give fewer TK+ colonies after reinfection with the ME111 virus than the parental BRL cells . However, we found that this is not the case . The TK transforming titer of the ME111 virus was the same (5 x 10" TK TU/mI of virus stock, data not shown) whether the titer was determined on the TK - ,NEOS cells or on the TK- ,NEO R cells that already contained one ME111 provirus . Thus the inhibition of expression of the second gene in the integrated proviruses of cells clones is a cis effect . Suppression of the Second Gene Is More Likely when the First Gene Is Transcribed by the LTR The data in Table 2 reveal that the amount of suppression of the genes in the ME 111 provirus is asymmetric . That is, the average amount of suppression of tk in Table 2 is greater than the average amount of suppression of neo in Table 2 . We wished to determine whether this asymmetry is a result of the nature of the selection for expression of each gene or of the promoter that transcribed these genes . Therefore, we repeated the experiments of Figure 2 and

BRL NEO

4,

R

CELL CLONE

SELECT FOR TK+ CELLS

*N

e

CHICKEN EMBRYO FIBROBLASTS CHRONICALLY INFECTED WITH REV-A

1 PEG FUSION

-

RECOVER LOW TITER VIRUS

INFECT D17 CELLS

- PICK NEO

R D17

COLONIES

RECOVER HIGH TITER VIRUS

~. TITER VIRUS FOR TK AND NEO TRANSFORMING ACTIVITY

Figure 5 . Protocol for Recovering and Testing the Biological Activity of Viruses from ME1 11 -Infected Rat Cell Clones Selection for the second gene means that TK' clones were tested for the NEO R phenotype and NEOR clones were tested for the TK' phenotype as described in the legend to Table 2 . The remainder of the cells were fused as described in Experimental Procedures . The low titer virus recovered from the fusion usually had a titer of about 1-50 transforming units per ml . The virus recovered by making producer clones of D17 cells by infection with the low titer virus had a titer of about 1 x 10' transforming units per ml . Virus titers were determined as described in Experimental Procedures,

Table 3 . Genetic Transforming Activities of Virus Recovered from Cell Clones Cells That Grew in NEO R BRL Cell Clone HAT'

Relative TK TU/NEO TU of Recovered Virus'

1

4

1

2

0 .1

1

<0 .01

1

3-6

Experimental protocol is diagrammed in Figure 5 . Cell clones were derived as outlined in Figure 2 . Each clone tested represents an independent cell clone . ' Cells were grown in G418 and then tested before fusion for plating efficiency in HAT . The percentage of cells that grow in HAT is the ratio of the plating efficiency of the cells in HAT divided by the plating efficiency of the cells in no selection times 100. (Cell clones here are the same as in the top of Table 2) NEO TU (transforming units) is the number of colonies per ml of virus stock that can be transformed to a NEO R phenotype . T K TU is the number of colonies per ml of virus stock that can be transformed from TK- to TK' phenotype. Numbers are relative to ME111 virus recovered from chicken embryo fibroblasts transfected with plasmid DNA (see Experimental Procedures) . ME111 virus gave a TK TU/NEO TU ratio of 1/24 ± 7 with NEO TU titered on D17 cells and TK TU titered on BRL TK - cells (see Experimental Procedures) . The titer of virus recovered from the cell clones all fell into this range . Duplicates of each determination were done . `This experiment was repeated with four independent cell clones, which all gave identical results.

Table 2 with virus recovered from the plasmid pME123 . In this virus (Figure 1), the tk gene is transcribed by the promoter in the LTR, while the neo gene product is translated from transcripts that begin in the tk promoter . Thus, relative to ME111, the promoter for each gene is switched . We infected BRL cells and selected the cells in either G418 or HAT . These clones were grown to 5 x 10 4 cells and tested for expression of the second gene in the provirus (tk after neo selection, neo after tk selection) . When cells were initially selected for tk expression, cell clones were

Cell 4 64

obtained that ranged in the percentage of cells that could subsequently grow in G418 from 10 to <0 .01% with an average value of 6% ± 2% (Table 2, bottom) . On the other hand, when cells were initially selected for neo expression, cell clones were obtained that ranged in the percentage of cells that could subsequently grow in HAT from 100% to 0 .1% with an average value of 28% ± 13% (Table 2) . Thus the average amounts of suppression are reversed in ME123 relative to ME111-the suppression of neo is greater than the suppression of tk . We conclude, therefore, that when the initial selection is for expression of the gene transcribed by the LTR-the stronger of the two promoters (Figure 1)-the second selectable gene is more likely to be suppressed than when the initial selection is for expression from the 3' and weaker promoter . Discussion We find that usually only one of two adjaent cistrons integrated into chromosomal DNA by a retrovirus vector is active, as defined by the ability of the infected cells to grow in selective medium . This conclusion comes from analysis of cell clones that harbor a single provirus containing two promoters and two genes . The promoter in the long terminal repeat is necessary for initial virus production and for expression of one of the selectable genes in the virus, while the tk promoter, which is placed internally in the viral genome, is necessary for expression of the second selectable gene in the virus . Transcription from the 5' promoter (LTR) will overlap with transcription from the 3' promoter (tk) . We find that in cell clones selected for expression of the 5' selectable gene, the 3' gene is usually suppressed, and that in cell clones selected for expression of the 3' gene, the 5' gene is usually suppressed (33 of 36 clones analyzed) . Inhibition of Proximal Promoters In a previous paper (Emerman and Temin, 1984), we suggested that expression from 5' promoters might inhibit expression from downstream promoters because we always found deletions that include a 5' promoter in recovered retrovirus vectors containing three promoters and one selectable gene . In the present case, with two promoters, we find that expression of one cistron is usually epigenetically suppressed . We hypothesize the expression from one promoter inhibits the expression from another nearby promoter . Thus suppression of one promoter (and hence the gene it expresses) is necessary for efficient expression from the other promoter . This suppression can occur genetically by deletions, as in our previous study with multiple internal promoters in the provirus, or by an epigenetic mechanism, as in the present study of proviruses with a single internal promoter . We also find in this analysis that cells that survive the second selection grow more slowly than cells grown in no selection (see Results) . Thus it is likely that degrees of expression occur. A biochemical analysis of the cell clones will allow us to define further the levels of suppression .

Preliminary evidence indicates that in cells infected with ME111 virus tk transcription is altered when TK activity is suppressed . Cullen et al . (1984) have recently described a phenomenon they attribute to inhibition of proximal promoters . They found that early after transfection the 3' LTR of an avian retrovirus DNA was prevented from transcribing a downstream gene when the 5' LTR was transcriptionally active . This phenomenon was called transcriptional interference . In our system, we find suppression of a 5' gene when the selection is for expression from the 3' promoter . In this case, the inhibition of expression cannot be ascribed to transcription from one promoter extending through the second promoter . We also find suppression of a 3' gene when we select for expression from the 5' promoter . This suppression could result from "transcriptional interference ." However, we also find clones in which both genes are active (Figure 3) ; therefore transcriptional interference does not appear to be involved . Epigenetic and Reversible Inactivation/Activation We were able to show that the suppression reported here is epigenetic because the second gene could be "activated" to a wild-type state when the provirus was released from the cellular genome by packaging of its RNA into virions, followed by reintegration of the viral DNA into other cell genomes . This result shows that the suppression of the gene is reversible and that the previous history of the provirus (i .e ., which of the two genes was previously inactivated) has no discernible bearing on the activity of that gene once it has been moved via infection to a new cell lineage . We have also shown that the activation state of the gene could be reversed without passage of the provirus through a viral stage because subpopulations of cells exist within the cell clones that could stably express both genes (Figure 2) . The clone-to-clone variation in the number of cells from each independent clone able to survive the second selection (Table 2) may be attributed to the activations of the second gene that occur during cell passage . The activation of the second gene is not reinfection of the cell population by the resident provirus, because although the BRL cell line produces insignificant amounts of virus (M . Emerman, personal observation), subpopulations with both genes active were also found in cell clones that had been established with helper-free virus that cannot replicate (data not shown ; Watanabe and Temin, 1983) . Suppression during cell passage also takes place . Figure 3, example II, illustrates one situation where cells from a cell clone that was initially selected for neo expression lost the ability to express the NEOR phenotype though they had gained the ability to express the TK+ phenotype . The Mechanism of Suppression Is Unknown We have shown that the suppression of one of the genes in the provirus is a cis effect . That is, it is a consequence of the surrounding DNA . However, how this effect is

Gene Suppression in Retrovirus Vectors 465

exerted is not known . The methylation of cytosine residues in cellular DNA has often been correlated with gene inactivation (Doerfler, 1983). We have looked for, but failed to find, methylation at the Hpa II, Sma I, Ava I, and Eco RI sites in activated genes (data not shown) . (It has been reported by Hardies et al . [1983] that methylation of the cytosine residue adjacent to the Eco RI recognition site in the tk gene can prevent cleavage at that site .) The site of integration might also influence the behavior of any particular provirus . For example, the development timing of activation of different integrated retroviruses has been attributed to their integration site (Jaenisch et al ., 1981) . We also find that the average amount of suppression of the second gene is greater when the first selection is for expression from the LTR (Table 2) . The LTR is the stronger of the two promoters (Figure 1) . This result may imply that the stronger promoter can inhibit expression of a nearby promoter to a greater extent than a weaker promoter . However, our results do not allow us to rule out the possibility that the degree of inhibition is also dependent on which promoter is 5' to the other . On the basis of all of these results, we propose a model in which transcription from one promoter causes a change in the chromatin structure in the surrounding DNA so that transcription from a nearby promoter is usually inhibited . This inhibition is not absolute and sometimes, perhaps because of secondary changes in the chromatin structure, transcription can efficiently occur from both promoters . Recovery of the provirus by passing it through the virion, followed by reintegration, frees it of its previous chromatin structure and allows a new one to be imposed . Experiments to test this model are underway . Transfection with Two Cistrons In contrast to our results, other investigators have transformed cells with selectable genes by transfection and found that linked genes with their own promoters are coordinately expressed (Girli et al ., 1983 ; Roginski et al ., 1983 ; Southern and Berg, 1982) . These results may differ from ours because in the transfection experiments stable transformed cells contain multiple copies of the transfected DNA . Thus the inhibition of a promoter in one gene may be compensated by the many copies of the genes in the cell . In our system, the cells contain only one copy of the provirus in their genome . Indeed, when cells that contain a provirus with one inactivated gene are reinfected to introduce a second copy of the provirus, the cells act phenotypically as if there was no inhibition of expression . Alternatively, expression from transfected DNA might be different than expression from a provirus . On the other hand, in a recent report Miller et al . (1984) have infected cells with retroviruses that contain both the HPRT gene and an inducible growth hormone gene . In some cell types they find a highly variable level of growth hormone production that is not due to mutations in the growth hormone gene . This may be a manifestation of the same phenomena that we describe .

Closely Linked Promoters in Normal Eucaryotic Cells A few systems have been described in which one cellular gene can be transcribed by two proximal promoters (Benyajati et al ., 1983 ; Kelly et al ., 1983 ; Schibler et al ., 1983) . However, since RNA is extracted from mixed populations of cells it is still not known whether both promoters are active at the same time in the same cell or whether some cells express a gene from one promoter, and other cells from the other promoter. Thus the generality of the phenomena of inhibition of expression of one promoter by expression of a closely linked promoter in eucaryotic cells is not yet known . However, it may be significant that in a number of cases the insertion of a transposon or retrovirus in a noncoding part of a gene can cause a mutant phenotype and that suppression of the mutant phenotype can occur by suppressing transcription from the inserted element (Jackson, 1984) . Thus it is possible that the initial mutation, like the inactivations described in this report, are the result of inhibition of expression of the normal gene as a result of transcription from a nearby promoter . Experimental Procedures Nomenclature TK', TK-, NEC', and NEOS refer to phenotype, while tk and neo refer to genes . Plasmids have a small p before their name (e .g ., pME111), while virus made from those plasmids does not (e .g ., ME111) . Cell clones with selectable proviruses in their genome are named with the cell type, followed by the virus name in parenthesis, e .g ., BRL (ME111), Cells Chicken embryo fibroblasts, Buffalo rat liver (BRL) cells, and D17 dog cells were grown as previously described (Emerman and Temin, 1984 ; Watanabe and Temin, 1983) . Selection for TK' cells was done in 10 -4 M hypoxanthine, 5 x 10-7 M methotrexate, and 3 x 10 -5 M thymidine (HAT) . Selection for G418-resistant cells (NEO R ) was done in 400 µg/ml G418 (Gibco) . Suppression is defined as inability of a cell that harbors the appropriate gene to grow in these conditions . Colonies surviving HAT selection and/or G418 section were picked in cloning cylinders ca . 1 or 2 weeks after infection . Plating efficiencies were determined by trypsinizing a dish of cells, counting the cells in a Coulter counter, and replating approximately 10 4, 10 3 , and 10 2 cells into 35 mm plates containing the appropriate selective medium . When cells were switched from HAT or any other selective or nonselective medium, 3 x 105 M thymidine and 10-4 hypoxanthine were always added to the medium for the initial week of growth . Virus Virus was recovered from plasmids as previously described (Emerman and Temin, 1984) . Virus titers were determined by infecting 2 x 10 5 cells in 60 mm tissue culture dishes with serial dilutions of the virus stock in the presence of 100 µg/ml polybrene in 0 .4 ml of medium . Selective medium was added 24 hr after infection . The TK transforming units or NEO transforming units of the virus stock is the number of phenotypically transformed cell colonies formed after infection multiplied by the dilution . Colony counts on duplicate plates varied by no more than 10% . The NEO transforming units of a virus stock were usually determined on D17 cells rather than on BRL TK- cells because BRL TK - cells are less sensitive to the drug G418 than D17 . When the neo gene is transcribed by the LTR the NEO transforming titers were about ten times higher on D17 (and chicken embryo fibroblasts) than on BRL cells . (Compare NEO TU for ME111 in Figure 1 [D17] to NEO TU of ME111 in Table 1 [BRL] .) This is presumably because the spleen necrosis virus LTR is a better promoter in dog and chicken cells than in rat cells (J . Embretson, personal communication) . The amount of transforming units of the virus stock was standardized

Cell 4 66

for variations in the amount of virus produced as follows : we infected chicken embryo fibroblasts with each virus stock and prepared DNA from the supernatant of a Hirt extraction (see below) from the infected cells three days later as previously described (Emerman and Temin, 1984) . The DNA was analyzed by electrophoresis in an agarose gel and bound to nitrocellulose. Parallel lanes were hybridized to either SNV-DNA sequences to determine the amount of helper virus in the stock or tk-specific DNA to determine the amount of recombinant virus . We quantified the hybridization in each lane by scanning the autoradiogram with a Beckman DU-8 scanning spectrophotometer. The relative virus production is the amount of recombinant virus as determined by hybridization, divided by the amount of helper virus, as determined by hybridization, relative to SW272 (see description below) . The specific transforming units of each virus stock is standardized by the relative virus production (transforming units/relative virus production) . Plasmid Constructions All DNA constructs were made by standard recombinant DNA techniques (Maniatis et al ., 1982) . The plasmid pSW272 has been previously described (Watanabe and Temin, 1983). It contains the tk gene with its own promoter but without its terminus within a deleted SNV genome. The plasmid pME111 was made as follows : the Xba I site of pSW272 was converted into a Hind III site (W. Gary Tarpley, personal communication) ; the neo gene was cut out of pSV2neo (Southern and Berg, 1982) with Hind III and Sma I ; Hind III linkers (P ._ L. Biochemicals) were ligated to the Sma I site, and the fragment was recut with Hind III, ligated into the Hind III site of pSW272, and characterized . The plasmid pME121 was made from pGT6 (Bandypadyhay and Temin, 1984b) by converting the Bam HI site immediately 3' to the tk gene into a Hind III site with S1 nuclease followed by ligation of Hind III linkers . The plasmids pME123 and pME124 were made in several steps . The DNA fragment containing the tk promoter from the Bam HI site to the Bgl II site of pTK A terR (Shimotohno and Temin, 1981) was ligated to a DNA fragment containing the neo gene from the Bgl II site to the Hind III site (formerly the Sma I site), the Barn HI site was converted to a Hind III site, and the fragment was subcloned into pBR322 . The Hind III fragment containing the tk promoter ligated to the coding sequences of the neo gene was then inserted into the Hind III site of pME121 . The plasmid pME123 differs from pME124 in the orientation of the insert . Recovery of Virus from Rat Cells Chicken embryo fibroblasts were chronically infected with REV-A, a wildtype virus closely related to the spleen necrosis virus from which the recombinants were constructed . Chronically infected chicken embryo fibroblasts (5 x 105) were plated on a 60 mm plate with 2 X 106 rat cells . Twenty-four hours later the medium (Eagle's medium with 5% calf serum) was removed, the cells were washed twice with medium that did not contain serum, and 2 ml of 45% PEG-8000 (Sigma) was added to the plate for exactly 2 min . The cells were then washed extensively and covered with 3 ml of Eagle's medium plus serum . Fresh medium was placed on the cells after 2 days, and medium was harvested on the third day after fusion . Nucleic Acid Analysis High molecular weight DNA was prepared as follows : tissue culture plates were washed twice with saline, lysed with 0 .6% SDS, 10 mM EDTA, and scraped into a tube . Proteinase K (Beckman) was added (100 µg/ml) for 12 hr at 37°C, followed by a phenol-chloroform extraction, and ethanol precipitation . The pellet was resuspended in 2 mM EDTA, treated with RNAase (50 ug/ml), treated with proteinase K (10 µg/ml), extracted with phenol twice and with chloroform once, and precipitated with ethanol again . Low molecular weight DNA was prepared by the method of Hirt (1967). Procedures for Southern transfer and hybridization were previously described (Emerman and Temin, 1984) . Acknowledgments We thank S . Hellenbrand and M . Newton for technical assistance, and C . Gross, R . Risser, W . Sugden, and members of our laboratory for helpful comments on the manuscript . This work was supported by Public Health Service grants CA-2243 and CA-07175 from the National Institutes of Health . M . E . is supported by Public Health Service training grant GM-07215 from the National Institutes of Health . H . M . T. is an American Cancer Society Research Professor .

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