Induction by HMBA and DMSO of Genes Introduced Into Mouse Eryth.Roleukemia and Other Cell Lines by Transient Transfection

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Induction by HMBA and DMSO of genes introduced into mouse eryth.roleukemia and other cell lines by transient transfection

Paul L. Campbell, Andreas E. Kulozik, Jean P. Woodham, and Richard W. Jones Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK

We have found rapid induction of various genes, including human globin genes, in response to hexamethylene bisacetamide (HMBA) and dimethyl sulfoxide (DMSO) in transiently transfected cells. In mouse erythroleukemia cells (MELCs), this effect is detected within 1 hr of exposure of the cells to inducer before the endogenous mouse globin genes are induced. It does not require protein synthesis and is reversed if the inducer is removed. This and other evidence suggest that the mechanism involves a change in activity of a factor intimately involved with transcription, probably as a result of post-translational modification. As such, it may represent an early triggering event in terminal differentiation, and its relevance to the expression of human globin genes in stable transfectants and to induction of the mouse globin genes is discussed. Other cell lines (K562 and NSO) also show this response, which may therefore involve a ubiquitous mechanism. We also found that HMBA depresses the expression of endogenous globin genes in K562, the opposite of this differentiation inducer's effect on MELC. [Key Words: Induction; differentiation; MEL; globin; HMBA] Received September 13, 1989; revised version accepted March 12, 1990.

Hexamethylene bisacetamide [HMBA} induces terminal of inducer, but after 8-12 hr, the cells reach a com- differentiation in a variety of cell lines {Marks and Rif- mitted state, in which terminal differentiation con- kind 19891. Of these, mouse erythroleukemia cells tinues, and the mouse globin genes are expressed in the (MELCs), a vitally transformed cell line that is blocked absence of inducer {Gusella et al. 1976; Chen et al. on the path to terminal erythroid differentiation, has at- 1982). tracted attention as a model for the study of both globin MELCs have provided a useful system for studies on gene expression and terminal differentiation {Friend et the regulation of human globin genes. Introduction of al. 1971; Marks and Rifkind 1989). The potential for ter- part of human chromosome 11 or all of 16 into MELCs minal differentiation is retained indefinitely by these leads to induced expression of the ~- or a-globin genes at cells and can be expressed by exposure to a variety of levels comparable with those of the endogenous mouse inducers. Among the changes in gene expression that ac- genes (Deisseroth and Hendrich 1979; Nandi et al. 1988). company this process is the induced transcription of the Stable transfectants containing the human a- or 13-genes mouse a- and 13m'J-globin genes (Profous-Juchelka et al. give low levels of expression, and although the 13-globin 1983; Ganguly and Skoultchi 1985), seen only after a lag gene is inducible, the a-globin gene is constitutive (Chao of between 12 and 24 hr (Salditt-Georgieff et al. 1984; et al. 1983; Wright et al. 1983, 1984; Charnay et al. Sheffery et al. 1984). The intervening time is occupied 1984). The more recent identification of a dominant by a complex series of events that may include changes control region (DCR} sited at the 5' end of the 13-globin that are prerequisites for globin gene expression. For ex- gene cluster {Grosveld et al. 1987; Ryan et al. 1989b; ample, induction is followed rapidly by changes in ino- Talbot et al. 1989; Tuan et al. 1989; van Assendelft et al. sitol metabolites (Faletto et al. 19851 and in the expres- 1989) has allowed high levels of human 13-globin expression of a number of proto-oncogenes {Ramsay et al. sion, equal to those of the endogenous mouse genes, 1986). Constitutive expression of one of these proto-on- when it is linked to the human gene in MELC stable cogenes, c-myc, can block terminal differentiation {Cop- transfectants {Talbot et al. 1989). Although the DCR pola and Cole 1986). In addition, cellular DNA un- provides a tissue-specific level of regulation, it is devel- dergoes a cyclical change in cytosine methylation (Razin opmental stage independent (Tuan et al. 19891, and et al. 19881. These early events occur at a time when the regulatory sequences closer to the globin genes may be differentiation process requires the continued presence responsible for stage-specific expression (Kollias et al.

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Rapid induction in transients

1986; Chada et al. 1987; Trudel et al. 1987). Regulatory Results sequences lying 5' and 3' close to the human 13 gene Dependence of expression on plasmid concentration have been identified in stable MELC transfectants and in transgenic mice (Behringer et al. 1987; Kollias et al. Preliminary experiments showed that the human a- and 1987; Trudel et al. 1987; Antoniou et al. 1988). The in- ~-globin genes could be rapidly induced following tran- volvement of both distant and local sequences in the sient transfection of MELCs. However, it was important regulation of the human a-globin genes remains more of to establish that their expression did not saturate and an enigma. However, the ability of the ~-globin cluster that induction did not vary over the range of plasmid DCR to support high levels of tissue-specific expression concentrations used for electroporation. of the a-globin gene in transgenic mice (Hanscombe et Uninduced MELCs were transfected with the human al. 1989; Ryan et al. 1989a) suggests the presence of an al-globin gene (pSPNaN) at a number of DNA concen- analogous sequence in the human a-globin gene cluster trations and grown with or without inducer (5 mM (Higgs et al. 1989). The question of involvement of se- HMBA) for 6 hr before harvesting (Fig. 1A). At input quences close to the a-globin genes remains more open. DNA concentrations where the human a-globin tran- We used electroporation to transfect MELCs transcript was detected, induction was associated with a 10- siently. Applied to the process of terminal differentia- to 20-fold increase in human transcript. As expected, tion, transient transfection may allow identification of there is no evidence for induction of the mouse a-globin individual events on the pathway to terminal differen- genes by 6 hr after exposure to HMBA (Salditt-Georgieff tiation and globin gene expression. We describe a rapid, et al. 1984; Sheffery et al. 1984). The accumulation of labile stimulation of transcription of a variety of appar- human ~ transcript was not related in a simple manner ently unrelated genes and artificial promoters following to the DNA concentration used for electroporation, and exposure to inducer. This is independent of protein syn- there was a threshold of 20-50 ~g/ml below which ei- thesis and is likely to involve post-translational modifi- ther transfection or expression appeared to be inhibited. cation. A similar result was obtained with the human 13-globin

Figure 1. Human globin gene expression following electroporation of MELCs at a number of plasmid concentrations. MELCs were electroporated in the presence of 5-200 ~g/rnl pSPNetN (A) or 10-200 ixg/ml pSPNI3N (B). Transfected cells were divided and grown with ( + ) or without ( - ) 5 mM HMBA for 6 hr (A) or 4 hr (B) before harvesting and RNA analysis. (A) Five micrograms of total RNA and samples of human IH) and mouse/Mu) bone marrow RNA hybridized with human IpSP6ct132) and mouse {pSP64AMet) et-globin probes gave the expected protected fragments, representing human (Ha) and mouse (M~t) a-globin mRNA. The molecular weight markers (M} in this and subsequent experiments are end-labeled HpaII-digested pBR322. Below is shown the genomic DNA insert containing the human cq-globin gene in pSPNotN. {B) Twenty-microgram samples of total RNA and a sample of human bone marrow RNA (H) were hybridized with a human f~-globin probe (pSP64H~142) to give 142- and 210-bp fragments protected by human ~ mRNA {H~). Below is shown the genomic DNA insert containing the human J3-globin gene in pSPN~N.

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Campbell et al. gene (pSPN13N; Fig. 1B). By 4 hr after exposure of trans- inhibit transfection or expression, the effect of cotransfected cells to inducer, the amount of human 13 tran- fecting with carrier DNA was examined {Fig. 2). MELCs script increased 10- to 20-fold over that in uninduced were transfected at concentrations of pSPNoLN ranging cells. As with the human a-globin gene, there was a from 2 to 100 ixg/ml, but with the total DNA concentra- threshold to expression between 20 and 50 ~g/ml of tion maintained at 100 ~g/ml by the inclusion of vector DNA. DNA [pSPNKN). Cells were transfected in duplicate at Because this threshold could be explained by either each pSPNaN concentration and grown in the presence specific or nonspecific binding of plasmid to sites that (Fig. 2B) or absence (Fig. 2AI of inducer. Radioactivity in

Figure 2. Human a-globin expression following electroporation of MELCs at different concentrations of pSPNaN in the presence of carrier DNA. (A and B} MELCs were electroporated at a constant plasmid concentration of 100 ixg/ml of which 2-100 ~g/ml was made up of pSPNaN and the remainder of pSPNKN (plasmid vector}. Duplicate transfections were performed for each pSPNaN concentration, and the transfected cells then divided and grown for 6 hr without (A) or with (B) 5 mM HMBA. Aliquots of 10 ~g (A) or 5 Ixg (B) of RNA were analyzed with pSP6a132 and pSP64AMa to give human a (Ha)- and mouse a {Mal-protected bands. (C and D) The radioactivities in human a RNA-protected bands were divided by those in mouse a RNA (Ha/Ma), and these ratios were plotted against the concentration of pSPNaN used for electroporation. The results of this for uninduced (C) and induced (D) ceils are shown. (E) The ratios (Ha/Mal in induced cells were divided by the corresponding ratios in uninduced cells to give induction ratios for each pSPNaN concentration. (F1 Total plasmid molarity (pSPNaN and pSPNKN) was calculated for each concentration of pSPNaN, and the ratio of human aRNA to mouse aRNA (Ha/Ma) was divided by this number to give a measure of pSPNaN expression per mole of plasmid, which was then plotted against pSPNaN concentration for induced transfectants. (G) MELCs were transfected as for A and B, but using pSPNaNAEco (5.8 kb) and plB8 (5.4 kb). Duplicate transfections were performed with 2 (lanes 1, 6), 8 (lanes 2, 7), 20 (lanes 3, 8 I, 50 (lanes 4, 9), and 100 (lanes 5, 10) txg/ml pSPNaNAEco, the total DNA concentration being maintained at 100 ixg/ml by the inclusion of pJB8. Shown is the analysis of 5 ~g of RNA for the presence of human (Ha) and mouse (Ma) a-globin transcripts in induced transfected cells. (H) The ratio Ha/Ma in G was plotted against pSPNaNAEco concentration.

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Campbell et al.

the protected human a RNA bands was expressed as a view of the lag of 12-24 hr characteristic of the response ratio to mouse a RNA for each DNA concentration (Fig. of the mouse globin genes. We therefore examined in 2C,DI. In both uninduced and induced cells, the amount more detail the accumulation of human transcript with of human a transcript increased linearly between 20 and time (Fig. 3). The mouse ~-globin genes responded to in- 50 izg/ml, but the system appeared to saturate above duction with a small increase in transcript detected by these values. However, induction is consistent 12 hr and an increase of 20-fold by 24 hr. The human throughout the range of pSPNaN concentrations used gene, on the other hand, gave a detectable response by 1 {Fig. 2El. Thus, provided carrier DNA was present, in- hr after induction, and the amount of R-globin transcript duction was seen at pSPNaN concentrations as low as 2 increased rapidly to a maximum between 3 and 6 hr be- Ixg/ml. At this concentration, the volume of an erythro- fore declining. The same decline in expression is seen leukemia cell would be occupied by -100 molecules of following transfection of MELCs with other genes (not this plasmid. Because the DNA is in solution and com- shown). plete equilibration of cell contents with the external me- The useful span of this transient assay is therefore dium is an unlikely consequence of electroporation, this limited to the first 3-6 hr following transfection. The figure is probably an upper limit for the copy number per subsequent decline in the amount of transcript may cell. have any of a number of causes, including cell death, If induction is an effect on transcription, then satura- destruction of plasmid, or inhibition of transcription. tion for ot-globin expression at similar concentrations of The stability of globin mRNA in MELCs (Volloch and pSPNaN in both uninduced and induced MELCs could Housman 1981) makes RNA degradation a less likely imply that induction does not affect binding of a rate- cause. We analyzed isolated nuclei for the presence of limiting factor but, rather, affects its ability to activate plasmid following transfection and found no significant transcription once bound. However, pSPNoLN is a 9-kb decrease in the amount of plasmid (not shown) and no plasmid, whereas the vector with which it was cotrans- evidence for the entry of plasmid DNA into nucleo- fected is 3 kb. Thus, although DNA concentration was somes (Enver et al. 1988). However, because we did not maintained at 100 lxg/ml, plasmid molarity declined as know what fraction of the DNA associated with the nu- pSPNaN made up an increasing proportion of the DNA. clei was transcriptionally active, we considered that ff the amount of oL-globin transcript in induced cells is these analyses would be difficult to interpret. corrected for total plasmid molarity and plotted against pSPNoLN concentration [Fig. 2F), the relationship is linear. The same is true for uninduced cells (not shown). Dependence of expression on the presence of inducer and on protein synthesis To test this relationship directly, cells were transfected with various concentrations of pSPNaNAEco (5.8 kb, de- The rapid response to induction of the transfecting rived from pSPNoLN and behaving indistinguishably globin genes could be due to either a stable or unstable from it; see Methodsl, together with plB8 {5.4 kb) so that both the total DNA concentration and plasmid molarity were approximately constant [Fig. 2G1. The amount of human oL transcript was then linearly related to pSPNotNAEco concentration {Fig. 2HI, in which case expression was directly proportional to the number of copies of the transfecting gene and the total molar concentration of plasmid. Therefore, this system does not saturate for either expression or induction when up to 100 ixg/ml of plasmid containing the a-globin gene is used for electroporation, provided carrier DNA is present. The dependence on molarity, whether of pSPNKN or the related cosmid vector plB8, would suggest that specific sequences in these plasmids affect either transfection or expression by binding with an inhib- itor. We have excluded the possibility that plasmid sequences contribute to induction by inserting the human a-globin gene in the NotI site of the unrelated vector pCos6EMBL (Ehrich et al. 1987; not shown}. Lineariza- tion of plasmids before transfection also does not affect Figure 3. Expression of the human a gene with time following induction {not shown I. transfection. MELCs were electroporated in the presence of 10 ag/ml pSPN~N and 90 wg/ml pSPNKN. The pool of transfected cells was divided and grown with (+) and without (-) 5 InM Time course of human a-globin expression following HMBA for the times shown. Five micrograms of RNA and induction samples of human (H) and mouse (Mu) bone marrow RNA were analyzed, by use of pSP6a132 and pSP64AMa, to give protected The rapidity with which the human genes were induced fragments representing human {Ha) and mouse (M~}-globin following transfection and induction was surprising in mRNA.

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Rapid induction in transients

change in the cells. To distinguish between these alter- sion of the transfecting genes, as well as the mouse natives, the effect on expression of removing inducer genes. To test this, uninduced cells and cells grown with from induced transfected cells was examined (Fig. 4A, HMBA for 36 hr were cotransfected with SPNoLN and lanes 1-4). Two aliquots of cells transfected with pSPhAPRT and then grown with or without HMBA for pSPNaN were grown for 2 hr in the presence of HMBA. an additional 6 hr {Fig. 4DI. The plasmid pSPhAPRT One was harvested iFig. 4A, lane 2), and the other was contains the hamster APRT gene, which is expressed grown for an additional 4 hr without inducer IFig. 4A, constitutively in both transiently transfected MELCs lane 3). Both of these cultures contained the same {shown here} and in stable MELC transfectants [Chao et amount of human tx RNA and half as much as a culture al. 1983). The human a-globin gene was expressed at a grown with inducer for the full 6 hr (Fig. 4A, lane 4J. similar level with respect to the APRT gene whether or Therefore, the induction response is reversed by removal not the cells had been exposed to HMBA before transfec- of HMBA. The same conclusion was drawn from analo- tion. A similar experiment showed the same to be true gous experiments with cells transfected with the human of the human ~-globin gene [Fig. 4D). Furthermore, irre- [3-globin gene. Thus, rapid induction of transient trans- spective of the time of exposure of the cells to inducer, fectants must be either a direct response to the inducer, sequences contained in the transfecting globin genes are which seems unlikely, or must involve an unstable in- not responsive to stable changes that allow the endoge- termediate. nous mouse genes to be expressed in the absence of in- When MELCs are exposed to inducer for 12 hr or ducer. longer, stable changes in the cells allow subsequent The rapid response of the human a-globin gene to in- mouse globin expression even if the inducer is removed duction and its lability suggested that de novo protein (Gusella et al. 1976; Chen et al. 19821. Longer exposures synthesis might not be required. Cells were transfected to inducer may therefore lead to commitment to expres- with pSPNaN, then divided and grown with or without

Figure 4. The dependence of induced expression of human globin genes on the presence of inducer, on protein synthesis, and on exposure of cells to HMBA prior to transfection. (AI MELCs were electroporated with 10 ~g/ml pSPNetN and 90 ~g/ml pSPNKN and grown without HMBA for 6 hr (lane 1 I, with 5 rn~ HMBA for 2 hr (lane 21, with 5 mM HMBA for 2 hr and, after harvesting, washing, and resuspension in fresh medium, for an additional 4 hr without HMBA {lane 3} or ior 6 hr with 5 mM HMBA (lane 4). Transfected cells were also grown without (lanes 5 and 7} or with (lanes 6 and 8} 5 mM HMBA and without (lanes 5 and 6) or with (lanes 7 and 81 1.5 ~g/ml cycloheximide for 6 hr. Five micrograms of RNA and samples of human (lane 9} and mouse {lane 10} marrow RNA were hybridized to give human {Ha)- and mouse (Ma)-protected fragments. {B) MELCs were transfected with 100 ~g/ml of pSPN13N and grown with {+ ) or without ( - ) 5 mbi HMBA and with (Cyc) or without 1.5 ~xg/ml cycloheximide for 6 hr. Twenty micrograms of RNA and an aliquot of human marrow RNA IH) were hybridized with a human 13 probe (pSP64HB1421, giving protected fragments representing the first and part of the second exon of human 13 mRNA (H131. [C) MELCs were transfected as in B, except that ceils were electroporated with 100 p.g/ml pSPN~/N, and 20-1xg samples of RNA and an aliquot of human marrow RNA (H) were hybridized with the human ~/probe (pSP65H~/1451 to give fragments protected by human ~/RNA (H~/). [DI Cells grown without inducer {0 hr} or with 5 m~ HMBA for 36 hr prior to transfection (36 hrl were electroporated with 75 ~g/ml pSPN13N and 75 ~g/ml pSPhAPRT {13J or with 10 ~g/ml pSPNaN, 75 ~g/ml pSPhAPRT, and 65 Ixg/ml pSPNKN(a). The cells were then grown for an additional 6 hr with ( + I or without { - I 5 mM HMBA. Ten-microgram samples of RNA were analyzed for the presence of human 13 mRNA {H131, and 5-~g RNA samples were analyzed for human et RNA IHal with pSP64H[3135, which protects most of the first exon of [3 mRNA, and pSP64cd32, respec- tively. Aliquots of 0.25 ~g RNA were analyzed for the presence of mouse et-globin mRNA (Met) with pSP64AMot, and 15-~g aliquots were analyzed for hamster APRT transcripts (hAPRT I. To correct for variation in transfection efficiency, radioactivities in human globin-protected fragments were divided by those in hAPRT. These ratios, for transfected cells grown with inducer, are shown below.

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Rapid induction in transients cycloheximide for 15 min before HMBA was added (Fig. pUCSV2cat contain the a- and f~-globin, and early SV40 4A, lanes 5-8). Cycloheximide did not abolish induc- promoters, pUCcat contains no eukaryotic promoter. tion, which therefore does not require protein synthesis. pIRV, which contains a 13-galactosidase gene driven by The human f~- and ~-globin genes also induced in the the rat f~-actin promoter (constructed by i. Morgenstern presence of cycloheximide (Fig. 4B, C). If proteins me- and H. Land, ICRF, London), was cotransfected with diate this induction response, they must do so by being each construct (Fig. 5B). Both pUCotcat and pUC~cat reversibly modified. were inducible, whereas pUCSV2cat was expressed constitutively. However, pIRV also responded to induction with a 5- to 10-fold increase in f~ogalactosidase activity. Specificity of induction Thus, on the one hand, the APRT gene (Fig. 4D) and a To explore the responsiveness of genes other than the number of viral promoters do not respond to induction, human globin genes, and to identify sequences that and on the other hand, o~-, ~-, and ~/-(not shown) globin confer inducibility, we used constructs in which the and rat f~-actin promoters do. We show below that the chloramphenicol acetyltransferase (CAT)gene is driven herpes simplex virus-1 IHSV-11 thymidine kinase (TK) by various viral promoters, or by the a- or ~-globin pro- gene promoter is also inducible. These results demon- moters (Fig. 5). pMMTVcat contains the mouse mam- strate that the induction response is not globin specific. mary tumor virus long terminal repeat (LTR} (J. Devine, unpubl.), and pSrM2cat contains the Moloney murine Cis-acting sequences responsible for induction sarcoma virus LTR (Laimins et al. 1982). pRSVcat contains the Rous sarcoma virus LTR (Gorman et al. 1982a}, To define further the c/s-acting sequences responsible pSV2cat contains the early promoter from SV40 for induction, a series of deletions were made in (Gorman et al. 1982b), and pAT153cat contains no eu- pUCacat by use of exonuclease III, and for deletions karyotic promoter. MELCs were transfected with each of close to the CAAT box, a series of oligonucleotides were these constructs, and one of three such experiments, all used to give deletions extending down to and through giving similar results, is shown (Fig. 5A). None of these the CAAT box. The response of these constructs fol- vitally promoted CAT genes gave significant increases lowing transfection and induction was measured by both in expression on induction, though in subsequent exper- CAT and RNA analyses (not shown). Deletion of se- iments, we detected a small and reproducible response quences from - 1375 to - 76 relative to the cap site gave by pRSVcat. The largest of any increase was seen when a decrease in the level of expression similar to that de- cells transfected with pMMTVcat, which contains a glu- scribed previously (Whitelaw et al. 1989), but induction cocorticoid responsive element (Lee et al. 1981), were remained unaffected. Removal of an additional 8 bp 3' to exposed to dexamethasone. position -76 destroyed the CAAT box and caused the Constructs in which the a- and f~-globin promoter re- loss of measurable expression. Induction must therefore gions replaced the early SV40 promoter in pSV2cat gave be mediated by sequences between an intact CAAT box inducible but weak responses in similar experiments. and the translation initiation codon. Higher levels of expression were consistently obtained To eliminate the possibility that sequences between when the plasmid vector sequence was replaced by the ot-globin cap site and the initiation codon are respon- pUC13 (see Methods). pUCotcat, pUCf~cat, and sible for induction, a universal leader sequence (uls), de-

Figure 5. Expression of virally promoted and et- and ~-globm-promoted CAT genes. (A) MELCs were transfected with 100 ~g/ml pMMTVcat (MMTV), pSrM2cat (SrM2), pAT153cat (AT153), pRSVcat (RSV), and pSV2cat {SV2). Transfected cells were grown for 12 hr, with ( + ) and without ( - ) 5 mM HMBA and, in one case, with 2 ~M dexamethasone (D). Shown below is the percent conversion of [14C]chloramphenicol to its major acetylated derivative per milligram of protein (%). (B) MELCs were transfected with 75 ~g/ml of plRV and 75 lag/ml of each of the following: pUCcat (pUC); pUCSV2cat (SV2); pUCacat {a); pUCBcat (13). Transfected cells were grown with {+ ) or without ( - ) 5 mM HMBA for 12 hr. Shown below is the percent conversion of [14C]chloramphenico1 per milligram of protein {%) and 13-galactosidase activity in milliunits per milligram (mU/mg) of protein.

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Campbell et al. signed to be linked to a number of inducible promoters, sequence around the cap site (Smale and Baltimore was inserted into pUCSV2cat. Into this construct the 1989). pUCI3ulscatA-79 is also inducible, but again its minimal promoter elements of the oL- and [3-globin genes expression is much reduced compared with pUC~catA- and the HSV-1 TK promoter were inserted [Fig. 6). These 371, which contains both the normal cap site and leader constructs preserve the normal spacing between the sequence and more sequence 5' to the CAAT box. TATA box and a potential new cap site. Expression of pUCTKulscat was also induced. Thus, inducibility is these constructs was less than that of those containing conferred by sequences lying between or including the the normal cap site and leader sequences, and this pre- CAAT and TATA boxes of the cx- and 13-globin genes and vented the use of RNase protection analysis. upstream from the cap site of the HSV-1 TK gene. When transfections were performed in the absence of Cotransfection of these constructs with pIRV affected plRV {Fig. 6A), the promoterless CAT control {pUCcat) induction (Fig. 6B). plRV, itself, gave an induction ratio gave low uninducible levels of CAT activity, whereas of -10-fold in all transfections. The more strongly ex- pUCSV2cat was expressed at a higher constitutive level pressed constructs, pUCoLcatA- 76 and pUCI3catA- 371, [Fig. 6A). These relative levels of expression were seen remained inducible in the presence of pIRV, though less consistently, pUCcxulscatA-76 lacks the normal cap so than in its absence. The ulscat constructs, pUCc~ul- site and leader sequence present in pUCoLcatA- 76 but scatA-76, pUC~3ulscatA-79, and pUCTKulscat, were still shows induction. However, its level of expression is then all constitutive, and the promoterless control con- reduced, probably as a result of changes to the normal struct, pUCcat, was inhibited on induction. This is pre-

human aloha-olobin ULS CAT

ell HII M[~ I Cep ~'- GCCAGCCAATG AGCGCCGCCC GGCCGGGCGT GCCCCCGCGC CCCAAGCATA AACCCTGGCG COCggccgac gcgtgectcc tagcttggcg ag

human beta-alobln ULS CAT

$pe i 8** H. Mlu P C*p CClctaGTTG GCCAATCTAC TCCCAGGAGC AGGGAGGGCA GGAGCCAGGG CTGGGCATAT AAGTCAGGGg cGcGCggccg acgcgtgac! cclag~tgg cgag

HSV-1 TK ULS CAT

Xho t r CCCGTGGCCC GTTGCTCGCG I"rTGGCTGGC GGTGTCCCCG GAAGAAATAT ATTTGCATGT CTTTAGTTCT ATGATGACAC AAACCCCGCC C,p Eco nl M;., ~,, HI: u.~, CAGCGTCTTG TCATTGGCGA ATTCGAACAC GCAGATGCAG TCGGGGCGGC GCGGTCCCAG GTCCACTTCG CATATTAAGG TGACGCGcgg ccgacgcglg aclcctagcl

Figure 6. Expression of constructs containing minimal promoter regions and a common cap site and leader sequence. (A) MELCs were transfected with 100 ~g/ml pUCcat, pUCcxulscatA- 76 (auls), pUC~ulscatA- 79 (~ulsl, pUCTKulscat (TKuls), pUCSV2cat (SV21, pUCc~catA - 76 (~tA- 76), or pUC~catA -371 (~A-371 ), and the transfected cells were grown with (+) or without {-) 5 mM HMBA for 18 hr. The percent conversion of [14C]chloramphenicol per milligram of protein was calculated for each sample, and the induction ratios for each transfection are shown below (ind. ratio). (BI MELCs were transfected as in A, except that 50 wg/ml of plRV was included in each transfection. Shown below are the induction ratios for CAT activity and [3-galactosidase activity in milliunits per milligram (mU/mgl of protein. Also shown are the sequences of the promoter regions of the uls CAT constructs. CAAT and TATA boxes and some restriction sites are underlined. Nucleotides corresponding to those in the intact genes are in uppercase letters.

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Rapid induction in transients

sumably due to competition imposed by the presence of pIRV, the effect being greatest on the most weakly expressed constructs.

Cell specificity of the induction response This induction response is not specific for globin genes, and to determine its cellular specificity, MELCs, K562, a human erythroid cell line, and NSO, a mouse myeloma line, were transfected with pUCacata-603 and grown with or without 5 mM HMBA or 1.4% dimethyl sulfoxide (DMSO). All three cell types showed a 10-fold induction of CAT with either inducer (not shown). Thus, the mechanism of induction is not cell-type specific. Although the ~- and [3-globin genes were both induced in transiently transfected MELCs, expression of the c~- globin gene was much greater than that of the [3-globin gene, a situation similar to that in nonerythroid cells (Banerji et al. 1981; Mellon et al. 1981; Humphries et al. Figure 7. Comparison of the induced expression of the a- and 1982; Treisman et al. 1983; Whitelaw et al. 1989). This B-globin genes and of Ig[3 in MELC and a mouse myeloma cell suggested that the transiently transfecting globin genes line NSO. MELCs [MEL) or NSO cells were cotransfected with gained no advantage from being expressed in an ery- 10 t~g/ml pSPNaN and either 50 t~g/ml pSPN[3N or 10 tLg/ml throid environment. Their expression was therefore IgB {see Methods}, the total DNA concentration being made to compared with that of a construct containing a well-de- 100 ~g/ml with pSPNKN. The transfected cells were then fined tissue-specific promoter and enhancer. Igf~ consists grown for 6 hr with {+ ) or without ( - ) 5 mM HMBA. 2.5 ~g (a, of an immunoglobulin gene promoter fused to the body Ig[3) or 12.5 ~g ([3) of RNA were analyzed. of the human [3-globin gene and including the IgH enhancer downstream (see Methods; K. Weston, unpubl.). This construct would be expected to express well in a fected K562 cells suggested that the endogenous globin myeloma cell line and poorly in MELCs (Banerji et al. genes might also respond. K562 cells were therefore 1983; Grosschedl and Baltimore 1985; Picard and grown with hemin, an inducer of K562 (Rutherford et al. Schaffner 1985). MELCs and NSO cells were cotrans- 1981), with HMBA, or with both inducers [Fig. 81. As fected with the oL-globin gene and either the ~-globin expected, heroin induced both the coglobin and the em- gene or Ig[3 (Fig. 7). To enable comparison of [3-globin bryonic r genes. However, HMBA caused a dra- gene expression with that of the other constructs, five matic reduction in the amount of both transcripts, irre- times as much pSPN~N was used for transfection and spective of the presence or absence of hemin. Visual in- five times as much RNA was analyzed so that the [3- spection of the cell pellets showed that the treated cells globin signal was multiplied 25 times relative to those of were virtually white compared with the yellow/orange the a-globin and Ig[3 genes. The a- and [3-globin genes color of untreated cells or the red of hemin-induced were induced in both cell lines and expressed at similar cells. Although growth was inhibited by HMBA, micros- levels relative to each other, implying that the expres- copy showed no evidence of extensive cell death. There- sion of the [3-globin gene, like that of the a-globin gene, fore, as well as stimulating expression of constructs in shows little regard for cell type in a transient expression transiently transfected K562, HMBA must also act at a system. This contrasts with the behavior of the Ig[3 gene regulatory step that is independent of that affected by in the same system. Its transcripts are undetectable in hemin. MELCs, compared with the a-globin gene, but it is expressed at a relatively high and nearly constitutive level in the myeloma cell line NSO, making its behavior com- Discussion parable to that of strongly expressed vitally promoted CAT constructs in MELCs {Fig. 5). The 5-kb BglII frag- We have shown that in transiently transfected MELCs, ment containing the human [3-globin gene that has been exposure to HMBA results in a rapid increase in expres- used in these experiments includes sequences impli- sion of the human ~-, [3-, and 7-globin genes and HSV-1 cated in tissue-specific expression in MELCs and trans- TK and rat [3-actin-promoted constructs. Although this genie mice (Charnay et al. 1984; Wright et al. 1984; response is not specific for globin genes, it does not ex- Townes et al. 1985; Behringer et al. 1987; Kollias et al. tend to strong retroviral or viral promoters or to the 1987; Antoniou et al. 1988). These regulatory elements hamster APRT gene and thus shows some template do not appear to confer on the ~-globin gene the high- specificity. Replacement of the normal transcribed se- level tissue-specific expression conferred by immuno- quences in several inducible genes does not prevent in- globulin regulatory sequences. duction, making changes in RNA processing, transport, The observation of induction in transiently trans- or stability unlikely as an explanation and leading us to

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Campbell et al.

step intimately involved in transcription initiation and is compatible with the findings of Cowie and Myers (1988), who used DEAE-dextran to transfect MELCs transiently with constructs containing the mouse [3- globin promoter. Mutations through this promoter affected expression and the extent of induction, but none abolished it. The labile nature of the induction response and the absence of a requirement for protein synthesis suggest that it may involve secondary modification of a transcription factor, for example, by O-glycosylation (Jackson and Tjian 1988) or phosphorylation (Sen and Baltimore 1986; Sorger et al. 1987; Sorger and Pelham 1988; Yamamoto et al. 1988; Bagchi et al. 1989; Hiebert et al. 1989; Raychaudhuri et al. 1989). RNA polymerase, which is known to be phosphorylated (Cisek and Corden Figure 8. Expression of the endogenous c~- and ~-globin genes 1989; Lee and Greenleaf 1989), would be among the can- in K562 cells following exposure to HMBA or hemin. K562 cells were grown without additions (lane 1) with 5 mM HMBA (lane didates for this role. Changes in protein kinase activity, 2) or 50 ~M heroin (lane 3), or with both inducers (lane 4) for 48 observed as an early event in MELC differentiation hr. Aliquots of 2.5 ~g of RNA were analyzed for the presence of (Marks and Rifkind 1989; Melloni et al. 1987), could c~, 4, and actin transcripts. The rat actin probe was also hybrid- provide the means of secondary modification, leading to ized in the absence of K562 RNA (S). increased binding of a factor intimately involved in transcript initiation. Constitutive promoters, such as those conclude that induction acts at the level of transcrip- of retroviral LTRs, may differ from inducible promoters tion. in not requiring this factor or they may not be rate-lim- The response depends on the continued presence of ited at the inducible step. Other early events in MELC inducer at both early and late times following induction. induction, such as changes in the expression of a Furthermore, pre-exposure of transfected cells to cyclo- number of proto-oncogenes (Ramsay et al. 1986) and a heximide shows that new protein synthesis is not re- cyclical change in DNA methylation (Razin et al. 1988), quired. These properties do not distinguish between a also do not require protein synthesis and may share a direct or indirect effect of inducer on transcription, but common transduction pathway with this induction re- neither HMBA nor DMSO affect in vitro expression of sponse. the human e~-globin gene in a MELC extract (P.L. Camp- Although HMBA causes induction of transiently bell and R.W. Jones, unpubl.); thus, we conclude that a transfecting genes in MEL, K562 and NSO cells, the cel- direct effect is unlikely. We note that in the case of the lular consequences of induction differ according to the human [3- and ~/-globin genes, but not the ot-globin gene, cell type. Although endogenous globin gene expression cycloheximide, by itself, causes an increase in the accu- is switched on in MELCs, it is switched off in K562 mulation of RNA and a superinduction when it is com- cells. Loss of erythroid and monocytic markers, with a bined with HMBA. This superinduction phenomenon concomitant increase in expression of megakaryocytic has been observed in a number of inducible systems markers, has been described following induction of K562 (Greenberg et al. 1986; Sen and Baltimore 1986). cells with phorbol 12-myristate 13-acetate (PMA)(Ta- The regions of the human a- and 13-globin promoters bilio et al. 1983; Siebert and Fukuda 1985; Alitalo et al. bounded by the CA_AT and TATA boxes (pUC~ul- 1988). The effect of HMBA on K562 cells may be similar scat&- 76 and pUC~ulscata-79; Fig. 6) are sufficient to that of PMA, and we speculate that the apparatus of for induction. More recently (P.L. Campbell and R.W. globin gene expression in K562 cells is unstable and that Jones, unpubl.), we detected induction of correctly initi- HMBA causes terminal differentiation at the expense of ated transcript from constructs lacking functional pro- globin expression down a nonerythroid pathway dictated moter sequences other than the TATA box and cap site by the cell. Many transformed cell lines undergo ter- upstream from the rabbit [3-globin structural gene and minal differentiation following exposure to HMBA the SV40 enhancer (Westin et al. 1987; Muller et al. (Marks and Rifkind 1989), and induction probably in- 1988). Thus, sequences upstream from the TATA box volves changes that are not specific to any one differen- may not be required. This possibility is strengthened by tiation program, whereas the program itself is largely a lack of correlation between upstream sequences and predetermined. The available evidence suggests that this inducibility. For example, the CAAT boxes of the induc- may be so for globin expression in MELCs. Thus, in ible HSV-1 TK promoter and the constitutive Moloney stably transfected cells, the DNase I-sensitive sites in murine sarcoma virus LTR bind the same protein the DCR show the same erythroid-specific pattern be- (Graves et al. 1986), and the HSV-1 TK promoter also fore and after induction (van Assendelft et al. 1989). shares functional SP1 sites (McKnight et al. 1984) with Also, the erythroid-specific protein-binding patterns the noninducible SV40 early promoter (Dynan and Tjian around the 13-globin gene and the abundance of the ery- 1983). This supports the view that induction acts at a throid transcription factor, GF-1, show little, if any,

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Rapid induction in transients change (de Boer et al. 1988; Wall et al. 1988; Tsai et al. A property of endogenous mouse globin gene induc- 1989). Furthermore, regulatory events determining tion is that it shows eventual commitment or inducer whether ~/- or ~-globin genes will express in MELC/ independence (Gusella et al. 1976; Chen et al. 1982). No human fetal erythroid cell hybrids occur before induc- data are available to determine whether the same is true tion (Papayannopoulou et al. 1986), showing that mech- of stably integrated inducible genes, but the transient re- anisms for regulating globin gene expression are already sponse does not show this property. Thus, other events in place. Consequently, globin gene expression in in the terminal differentiation process would be required MELCs may be blocked at steps that are not erythroid to explain commitment. specific. The rapid induction response that we have described Changes detected in transient transfectants as a non- is of interest from several points of view. We provide specific increase in the activity of the transcription ap- evidence that the activity of a ubiquitous component of paratus could provide a positive component in the acti- the cellular transcription apparatus is regulated by sec- vation of cellular and stably integrated genes during ondary modification. It is likely that its abnormal regu- MELC differentiation. If so, then one expectation would lation by HMBA or DMSO has a normal counterpart be of a correlation between the behavior of genes in that has a role in cellular regulation of transcription. transient and stable MELC transfectants. In stably Loss of this normal control may be related to the trans- transfected MELCs, the ~- and ~-globin genes and the formed phenotype and may contribute to the block on HSV-1 TK promoter have all been observed to induce, terminal differentiation. Thus, elucidation of this pro- whereas the APRT gene is expressed constitutively cess may contribute to an understanding of the trans- (Chao et al. 1983; Wright et al. 1983; Charnay and Henry formed cell phenotype, the requirements of the terminal 1986). These observations correlate with the behavior of differentiation process, and, in the case of MELCs, these genes in transiently transfected MELCs. On the globin gene expression. To do this, we are attempting to other hand, constitutive expression of the human c~- identify the factor modified by induction by using an in globin gene in stable transfectants (Charnay et al. 1984) vitro transcription system. appears to be at odds with its induction in transients. However, constitutive expression only implies that the rate-limiting step is constitutive and does not exclude Methods the presence of an inducible step that may be rate-limiting in other circumstances, for example, in transient Cell culture transfectants or when the gene is linked to a DCR. The MELCs 707 B10/1 were grown in suspension in RPMI 1640 me- variability in the induction response seen with stably dium (GIBCO), supplemented with 10% fetal calf serum, gluta- integrated genes (Wright et al. 1983, 1984; Charnay et al. mine (2 m~), penicillin (50 U/ml), and streptomycin (50 ~g/ml). 1984) appears to be a function of the integration site, an To induce differentiation, HMBA (Sigma) was added to the me- effect that is abolished when the gene is linked to the dium at a final concentration of 5 mM. Alternatively, 1.4% DMSO was used. Cycloheximide was used at a concentration of ~-globin DCR {van Assendelft et al. 1989). The effects on 1.5 ~g/ml, sufficient to inhibit protein synthesis in MELCs by induction of both integration sites and globin local regu- >95% (Kaneda et al. 1985), and dexamethasone at 2 ~M. A non- latory sequences (Antoniou et al. 1988) may be due to secretory mouse myeloma cell line (NSO) and the human ery- their binding of inducible factors, but these sequences throid cell line (K562) were grown under the same conditions as may also determine whether or not a single inducible MELCs. step acting elsewhere is rate-limiting and therefore measured. Trans[ection Induction of stable transfecting genes and the mouse globin genes is delayed for 12-24 hr following exposure MELCs were transfected by electroporation (Boggs et al. 1986; to inducer (Salditt-Georgieff et al. 1984; Sheffery et al. Chu et al. 1987). Cells were grown to a density of between 0.5 • 106 and 1.0 • 106/mI. They were harvested, washed once 1984). This delay indicates that the early stimulation of with PBS, and resuspended in 1 ml of PBS containing, usually, transcription seen in transient transfectants is not suffi- 100 I~g of supercoiled plasmid DNA. The cell suspension was cient to cause increased expression of chromosomally put into a 4-ml capacity plastic cuvette, into which were in- integrated genes. This could be due to a requirement for serted two flat electrodes facing each other, 8 mm apart. A 1000 de novo synthesis of transcription factors. Alternatively, ~F capacitor array, charged at 425 volts from an LKB 2197 the delay may be a function of chromosomal integration. power supply, was discharged through the cells. After electro- For example, evidence exists that access of protein poration, the cells were resuspended in medium and aliquots factors to DNA is inhibited by incorporation of DNA were distributed between flasks to which any additions had al- into chromatin (Emerson and Felsenfeld 1984; Workman ready been made. The whole procedure was conducted at room and Roeder 1987~ Workman et al. 1988; Cheng and Kelly temperature. NSO and K562 cells were electroporated in the same way, except that 400 volts was used. 1989), and this would not apply to naked DNA intro- Where necessary, a constitutively expressed plasmid duced into the cell by transient transfection. Thus, pro- (pSPhAPRT) was included as a control for transfection effi- teins modified by induction may depend for access on ciency. Alternatively, variation in transfection efficiency was cell-cycle events (Conkie et al. 1981), replication {Enver allowed for by duplication of the transfection. Transfection ef- et al. 1988), or changes in DNA methylation (Razin et al. ficiency for one batch of cells generally varied within a two- to 1988). threefold range. Cells (4 x 10z) transfected and grown for

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Campbell et al. various times provided between 50 and 100 ~g of total RNA, Constructs with a common cap site and leader sequence and multiples of this number of cells were electroporated to A 31-mer oligonucleotide, 5'-CTAGAGGGCGCGCGGCC- provide sufficient transfected cells for the number of experi- GACGCGTGACTCCT-3', was annealed with a complemen- mental conditions. tary 27-mer to give a double-stranded oligonucleotide with a blunt 3' end and a SpeI complementary 5' overhang. It also con- Constructs used for transfection tained BssHII, EagI, and MluI sites to enable different promoters to be inserted, preserving the distance between the The polylinker sequence of pSP65 was altered by use of stan- TATA box and the new potential cap site. The HindIII site in dard methods to give the following sequence of restriction the pUC13 polylinkei 3' to the CAT gene in pUCe~catA-76 sites: EcoRI, SacI, SpeI, SacII, SfiI, NotI, BamHI, KpnI, BamHI, was cut, filled in using Klenow polymerase, and religated. The XhoI, NotI, PstI, and HindIII. Genomic DNA fragments con- resulting plasmid was cut at the HindIII site at the junction of taining globin genes were inserted into this vector (pSPNKN) in the ~ promoter and the CAT gene, the ends were filled, and the the same orientation, the 5' end of the gene being nearest the plasmid was cut with BssHII. The double-stranded oligonucleo- EcoRI site. A 6.0-kb BglII-XhoI fragment containing the human tide (above) was cut with BssHII, and the resulting BssHII/blunt eq-globin gene from ca3' Bg (Nicholls et al. 1987) was inserted fragment was inserted between the BssHII/blunt HindIII sites between the BamHI and XhoI sites of pSPNKN to give of pUCacatA - 76 to give pUCaulscatA- 76. pSPNaN. Deletion between the EcoRI and XhoI sites 3' to the A double-stranded oligonucleotide, spanning the CAAT and gene in pSPN~N gave pSPN~NAEco. Insertion of a 5-kb BgllI TATA boxes of the [3-globin promoter, was made: fragment containing the human [3-globin gene into the BamHI 5'- CCACTAGTTGGCCAATCTACTCCCAGGAGCAGGG- site of pSPNKN gave pSPN~N, pSPN~N was made by inser- AGGGCAGGAGCCAGGGC TGGGCATATAAG TCAGGG- tion of the 3.3-kb HindIII fragment containing the human &/ GCGCGCCA-3'. This was cut with SpeI and BssHII, and the gene into the BamHI site of pSPNKN, pSPhAPRT was made by resulting fragment was inserted between the equivalent sites in transfer of the hamster APRT gene in a 7.8-kb HindIII fragment pUC~ulscatA - 76 to give pUCj3ulscatA- 79. (Lowy et al. 1980) into the HindlII site of pSPNKN. pUCTKulscat was made by insertion of the promoter region Ig~ is pV473[3GTIg3 [a gift from K. Weston (unpubl.)]. It con- of the HSV-1 TK gene into pUCemlscatA- 76. A 2-kb XhoI-lin- sists of the mouse VH promoter from pSV-V~I (Neuberger 1983) kered PvuII fragment containing the HSV-1 TK gene (McKnight fused to the human [3-globin structural gene with the IgH en- 1980) was made blunt-ended, by use of Klenow DNA poly- hancer inserted 3'. merase, and cut with MluI. The 190-bp M1uI/blunt fragment containing the HSV-1 TK promoter region was inserted be- Globin-promoted CA T constructs tween the SpeI site (filled) and the BssHII site of pUComl- pUC~cat and pUC[3cat were made as follows. The NcoI sites in scatA-76 to give pUCTKulscat. pSPNaN and pSPN~N were cut and made flush using mung bean nuclease, and a HindlII linker was inserted. EcoRI-HindIII Plasmid growth and preparation fragments containing the respective promoter regions were inserted between the AccI and HindIII site of pSV2cat (Gorman et All plasmids were propagated by transformation of Escherichia al. 1982a) after the EcoRI and AccI ends had been filled by use of coli RRI. Plasmid DNA was prepared by alkaline SDS lysis and Klenow DNA polymerase. The SacI-BamHI fragments con- purified by CsC1 equilibrium centrifugation (Maniatis et al. taining a globin promoter contiguous with the CAT structural 1982). gene were transferred to the same sites in the pUC13 polylinker to give pUCc, cat and pUC[3cat, pUCSV2cat was made by cut- RNA preparation ting of pSV2cat {Gorman et al. 1982b) with AccI, filling of the ends with Klenow DNA polymerase, and transfer of an AccI- A composite of existing methods was used {Auffray and Rou- HindIII fragment between a filled SpeI and a HindIII site of pU- geon 1980; Maniatis et al. 1982). MELCs were harvested, C~cat, which thereby replaced the ~x-globin promoter with the washed once with PBS, and homogenized in 10 ml of 6 M urea, 3 SV40 early promoter, pUC~catA- 371 was made by deletion be- M LiC1 for 1 min. After standing at 4~ overnight, the RNA was tween the NotI site 5' to the promoter region in pUC[3cat and pelleted at 10,000 rpm in a Sorvall SS34 rotor. The pellet was an AccI site at position -371. dissolved in 0.5 ml of 6 M guanidinium thiocyanate, 10 mM Tris (pH 7.4), 0.5% Sarkosyl, 200 mg of CsC1 dissolved in this solution, and 0.5 ml layered on 0.5 ml of CsC1, 1.7 g/ml, in 10 n~ 5' Deletion of the a-globin promoter region Tris (pH 7.4), and 10 mM EDTA. After centrifugation at 85,000 pUCacatA- 603 was made by deletion between the SpeI site of rpm in the Beckman TLA 100.2 rotor for 4.5 hr at 20~ the the 5' polylinker and a SpeI site at -603 in pUCacat. Exonu- supematant was removed and the RNA was dissolved in 0.4 ml clease deletions were made by use of the SpeI and SacI sites 5' of 10 mM Tris (pH 7.4), 10 mM EDTA, and 0.1% SDS. The RNA to the promoter of pUCacatA- 603 (Hoheisel and Pohl 1986). was ethanol-precipitated and redissolved in 80% formamide hy- Oligonucleotide-constructed a-globin promoter deletions bridization buffer. The RNA was quantitated by measurement were also made. Single-stranded oligonucleotides corre- of the E26o of a diluted aliquot. sponding to the antisense strand of the a-globin promoter extended from the EagI site at -56 from the cap site to positions RNA analysis at and between - 68 and - 112 from the cap site. The 3' ends of these were abutted to a SpeI site and extended an additional 6 RNA was analyzed by RNase protection with the SP6 system bp to form a terminal 18-bp palindrome. These oligonucleotides (Melton et al. 19841. RNA recovery was monitored either by were made double-stranded by mutually primed synthesis (Hill observation of cross-hybridization of the human probe to et al. 1986), the products were digested with SpeI and EagI, and mouse sequences or by inclusion of a probe to cell RNA. Uni- these fragments were inserted between the SpeI and EagI sites formly labeled KNA probes were made by use of [cz-a2p]GTP of pUCacatA- 603 to give pUCacatA- 68 to A- 112 inclusive. (>400 Ci/mmole, Amersham). RNA-RNA hybridization, with

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Rapid induction in transients

5 x l0 s cpm of radiolabeled probe and 0.25-20 ~g of total cel- Acknowledgments lular RNA, was performed at 50~ for 18 hr. RNase digestion was carried out at 21~ for 30 min. The protected RNA frag- We thank David Conkie for the MELC line 707 B10/1 and Alain ments were run on a denaturing 8% or 12% polyacrylamide gel. Townsend for the mouse myeloma cell line NSO. We also End-labeled HpaII-cut pBR322 was used as molecular weight thank Tom Maniatis /or pSP6Hcd32 and pSP64M~, Bemie marker. Fuji RX medical X-ray film was exposed to the dried Morley for pSP64H[3135, John Bennett for pSP64H~110, Peter gels at -70~ with an intensifying screen. Quantitation was Ratcliffe for the rat actin probe, Doug Higgs for Cc~3'Bg, Richard done by cutting out the radioactive bands, together with appro- Axel for the hamster APRT gene, Kathleen Weston for priate backgrounds, and the radioactivity was measured by pV473f3GTlg3, Joan Devine for the virally promoted CAT con- scintillation counting with an LKB-Wallac Beta-plate counter. structs, and J. Morgenstem for pIRV. We also thank Walter Schaffner for his helpful suggestions and the OVEC series of plasmids. We are also grateful to Gerry Warner and Roger SP6 probes Belcher for their considerable help with the design and con- The 5' human a probe pSP6cd32 (Chamay et al. 1984} and the struction of the electroporation apparatus and to Linda Roberts 5' mouse et probe pSP64M~ (Baron and Maniatis 1986) were and Liz Rose for typing the manuscript. This work was sup- gifts from Tom Maniatis. We used a derivative of pSP64Me, ported by the Medical Research Council (MRC). P.L.C. is in re- (pSP64AM~), made by deletion between a Bali site in the first ceipt of a MRC training fellowship. exon and a mung bean nuclease-treated EcoRI site in the poly- The publication costs of this article were defrayed in part by linker sequence. This is protected by a 91-bp fragment from payment of page charges. This article must therefore be hereby mouse ~-globin mRNA. Two 5' human f~ probes were used. marked "advertisement" in accordance with 18 USC section One a 555-bp BamHI-BalI fragment extending from +476 to 1734 solely to indicate this fact. -79 of the B-globin gene and cloned into pSP64 (pSP64H[~142; Kulozik et al. 1988), is protected by 142- and 210-bp fragments References of the first and second exons of BmRNA. The other, a gift from Bernie Morley, is a 226-bp BstNI fragment protected by 135 bp Alitalo, R., T.P. Makela, P. Koskinen, L.C. Andersson, and K. of the first exon of the human ~ gene (pSP64HB135). The 5' Alitalo. 1988. Enhanced expression of transforming growth human ~/ probe is a 1502-bp BstEII-HindIU DNA fragment factor [3 during megakaryoblastic differentiation of K562 from + 159 to -1343 of the human ~ globin gene cloned in leukemia cells. Blood 71: 899-906. pSP65, giving a 145-bp protected fragment representing the first Antoniou, M., E. deBoer, G. Habets, and F. Grosveld. 1988. The exon of the a~/gene (pSP65H~145). The human ~ probe contains human B-globin gene contains multiple regulatory regions: a 662-bp EcoRI-BglII fragment, overlapping the 5' end of the Identification of one promoter and two downstream en- ~-globin gene and inserted in pSP64, to give a 110-bp protected hancers. EMBO J. 7: 377-384. fragment. The 5' hamster APRT probe contains a 862-bp Auffray, C. and F. Rougeon. 1980. Purification of mouse immu- HindII-BamHI fragment overlapping the 5' end of the hamster noglobulin heavy-chain messenger RNAs from total my- APRT gene {Lowy et al. 1980) inserted between the HindlI and eloma tumor RNA. Eur. J. Biochem. 107: 303-314. BamHI sites of pSP64 (pSP64APRT}. The actin probe contains a Bagchi, S., P. Raychaudhuri, and J.R. Nevins. 1989. Phosphory- 276-bp AvaI-HindIII fragment inserted in pAM19, which pro- lation-dependent activation of the adenovirus-inducible E2F tected the 76-nucleotide first exon of the rat ~-actin transcript transcription factor in a cell-free system. Proc. Natl. Acad. (Nudel et al. 1983). Cross-hybridization of this probe to human Sci. 86: 4352-4356. RNA was used as a control for RNA recovery. The 5' cate~ Banerji, J., S. Rusconi, and W. Schaffner. 1981. Expression of a probe was made by inserting the SpeI-PvuII fragment of pU- globin gene is enhanced by remote SV40 sequences. Cell C~catA-112 between the XbaI and HindII sites of pSP64 27: 299-302. (pSP64~cat). Banerji, J., L. Olson, and W. Schaffner. 1983. A lymphocyte-specific cellular enhancer is located downstream of the joining CAT assay region in immunoglobulin heavy chain genes. Cell 33: 729- 740. MELCs transfected with constructs containing the CAT gene Baron, M.H. and T. Maniatis. 1986. Rapid reprogramming of were grown for 8-20 hr, harvested, and washed once with PBS. globin gene expression in transient heterokaryons. Cell The cells were resuspended in 200 vd of 0.25 M Tris (pH 7.4) and 46: 591-602. subjected to three cycles of freezing and thawing. Cell debris Behringer, R.R., R.E. Hammer, R.L. Brinster, R.D. Palmiter, and was removed by centrifugation, and aliquots of the supernatant, T.M. Townes. 1987. Two 3' sequences direct adult ery- containing between 100 and 500 ~g of protein, were used for throid-specific expression of human f3-globin genes in trans- the assay of CAT activity (Gorman et al. 1982b). For each assay, genic mice. Proc. Natl. Acad. Sci. 84: 7056-7060. 0.5 wCi of [14C]chloramphenicol (50 mCi/mmole, Amersham) Boggs, S.S., R.G. Gregg, N. Borenstein, and O. Smithies. 1986. was used. Following chromatograpy, the TLC plate was autora- Efficient transformation and frequent single-site, single- diographed overnight, the positions of [x4C]chloramphenicol copy insertion of DNA can be obtained in mouse erythroleu- and its acetylated derivatives identified, cut from the plate, and kemia cells transformed by electroporation. Exp. Haematol. their radioactivity measured by scintillation counting. The pro- 14: 988-994. tein content of cell extracts was measured by use of the Brad- Bradford, M.M. 1976. A rapid and sensitive method for the ford colorimetric assay (Bradford 1976). quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72: 248- ~-Galactosidase assay 254. ~-Galactosidase was assayed by use of o-nitrophenyl-~-D galac- Chada, K., J. Magram, and F. Costantini. 1987. An embryonic topyranoside (Herbomel et al. 1984). ~-Galactosidase {Sigma) pattern of expression of a human foetal globin gene in trans- was used as a standard, and activities were calculated as mil- genic mice. Nature 319: 685-689. liunits per milligram of protein. Chao, M.V., P. Mellon, P. Chamay, T. Maniatis, and R. Axel.

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CampbeHet~.

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Induction by HMBA and DMSO of genes introduced into mouse erythroleukemia and other cell lines by transient transfection.

P L Campbell, A E Kulozik, J P Woodham, et al.

Genes Dev. 1990, 4: Access the most recent version at doi:10.1101/gad.4.7.1252

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