MOLECULAR AND CELLULAR BIOLOGY, July 1990, p. 3838-38420270-7306/90/073838-05$02.00/0Copyright © 1990, American Society for Microbiology

Synergy between the NF-E1 Erythroid-Specific Transcription Factorand the CACCC Factor in the Erythroid-Specific Promoter of the

Human Porphobilinogen Deaminase GeneJ. FRAMPTON,t M. WALKER, M. PLUMB, AND P. R. HARRISON*

Cancer Research Campaign Beatson Laboratories, Beatson Institute for Cancer Research, Garscube Estate,Switchback Road, Bearsden, Glasgow G61 IBD, Scotland

Received 7 February 1990/Accepted 16 April 1990

A 114-base-pair promoter fragment of the human porphobilinogen deaminase gene functioned in anerythroid-specific manner in transient transfection experiments. Site-directed mutagenesis of the binding sitefor the erythroid-specific transcription factor (NF-E1) or an adjacent CACCC motif abolished the promoteractivity. Increasing the spacing between these sites progressively reduced promoter activity, but there was no

evidence that a critical alignment of the two factors on the DNA helix was required.

The promoters or enhancers of a variety of chicken,human, and mouse globin genes have been found to containbinding sites for a transcription factor (NF-E1) that has beenshown to be essential for erythroid-specific expression (5-7,14, 15, 17, 18, 23, 25). NF-E1 has also been implicated in theerythroid-specific transcription of the gene encoding theheme pathway enzyme, porphobilinogen deaminase (PBG-D) (11, 12, 15). In these various genes, the NF-E1-bindingsite is found in different contexts with respect to the prox-imity of binding sites for other transcription factors such asNF1, Spl, and the CAAT and CACCC factors, and it isknown that a single NF-E1-binding site does not function inisolation (15, 18); this finding suggests that NF-E1 cooper-ates with the factors binding to these other sites in order tofunction.

In the case of the PBG-D promoter, the human and mousesequences are highly conserved and contain an adjacentNF-E1-binding site and a CACCC motif, but the CACCCmotif occurs as a tandem duplication in the mouse genome(1). (The human PBG-D [hPBG-D] promoter also contains abinding site for another erythroid-specific factor, NF-E2, atabout -180 nucleotides [nt] [11, 12], but this site is notconserved in the mouse promoter [1].) Using as a model a

minimal 114-base-pair (bp) truncated hPBG-D promoter thatretains erythroid-specific activity, we have examinedwhether interactions between NF-E1 and an adjacentCACCC motif are essential for the erythroid-specific activityof the hPBG-D promoter.Two fragments of the hPBG-D 5'-flanking region between

-114 and +76 bp or between -700 and +76 bp were clonedupstream of the bacterial chloramphenicol acetyltransferase(CAT) gene [plasmids pPBG(-114)CAT and pPBG(-700)CAT; Fig. 1]; the 114 bp of 5' promoter sequence present inpPBG(-114)CAT contains binding sites for NF-E1 (CCTTATC, at -70 bp, reading the coding strand) (11, 15) and theCACCC factor (at -100 bp), as well as a protein-binding siteat about -30 bp. Both constructs were assayed by transienttransfection into either MEL cells or STO fibroblasts asfollows. One day before transfection, 2 x 106 cells wereplated into 80-cm2 dishes and then transfected with 5 ,ug of

* Corresponding author.t Present address: European Molecular Biology Laboratory,

Meyerhofstrasse 1, 6900 Heidelberg, Federal Republic of Germany.

CAT plasmid together with 5 ,ug of a plasmid containing theP-galactosidase gene driven by the IE-4 promoter of herpessimplex virus type 2 (pHSVpgal; for normalization of trans-fection efficiency [13]) and 10 ,ug of carrier DNA. Collectionof cells and assay for CAT activity were performed asdescribed previously (15). pPBG(-114)CAT gave a fivefold-increased CAT activity in MEL cells compared with STOcells, with a slightly larger erythroid-specific effect with thelarger promoter construct pPBG(-700)CAT (Table 1).To study in more detail the role of NF-E1 in regulating the

hPBG-D promoter, in particular whether it cooperates withthe factor binding to the adjacent CACCC motif, the effectsof mutating or altering the spacing of the NF-E1-binding siteand the CACCC motifwere examined. To facilitate measure-ment of hPBG-D promoter activity at the RNA level intransiently transfected MEL cells, the hPBG-D promotersequence from -114 to +76 bp or mutated versions of itwere cloned upstream of the human growth hormone (hGH)gene (since hGH mRNA is considerably more stable thanCAT mRNA in MEL cells). MEL cells were transfected withthe hGH gene constructs as described previously for theCAT gene constructs, and hGH assays were carried out 36 hafter transfection with 1 ml of medium, using the AllegrohGH radioimmunoassay kit (Biogenesis Ltd.). After the cellswere harvested, 1/10 was used for the 3-galactoside assay (8)and the rest was used for preparation ofRNA by the RNAzolmethod (3). The levels of hGH produced were then normal-ized with respect to a standard p-galactosidase activity.Either of two mutations that we have previously shown toaffect NF-E1 binding (15) (CCTTATC to AATTCTC or CC

TABLE 1. Expression of hPBG-D-CAT gene constructstransfected into erythroid and nonerythroid cell types

CAT activityaConstruct

STO cells MEL cells Ratio

PaCATOb 1 1 1pPBG(-700)CAT 0.4 3.8 9.5pPBG(-114)CAT 1.0 5.3 5.3

a Average of two independent transfections, with CAT activities differingonly by about 10%.

b Contains a minimal 52-bp mouse a-globin promoter upstream of the CATgene in p22 and was used as a control, since it has been shown not to have anyerythroid specificity (15).

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TABLE 2. Effect of mutations in the NF-E1-binding site andCACCC motif on hPBG-D promoter activity

Construct hGH releasea

Wild-type (pPBGhGH1) ......................................... 100NF-E1 site mutationsCCTTATC--CCTCATC ...................................... 5 ± 2CCTTATC--AATTCTC ...................................... 4 ± 1

CACCC mutationsCACCC-*GGGCC ........................................... 12+ 2Deletion mutant .......................................... 18 ± 5

a Percentage of wild-type value (- standard error) after subtraction of thevalue for the promoterless hGH vector (pOGH).

TCATC) reduced the hPBG-D promoter activit, almostcompletely (to about 12% of the wild-type activity, com-pared with 5% for the promoterless hGH vector alone);mutating the CACCC motif to GGGCC or deleting a 24-bpfragment containing the CACCC motif had a similar effect(Table 2). Both the NF-El-binding site and the CACCCmotif are therefore essential for promoter activity.

Insertion of a 71-bp fragment of polylinker sequence intothe Ball site between the NF-E1-binding site and the

A-700 +76

-114 +76

CACCC motif in pPBGhGH1 to give pPBGhGH1+71 (Fig.1) was found to reduce hGH production in MEL cellseightfold. Si protection analysis of RNA extracted fromMEL cells 36 h after transfection with pPBGhGH1 by usingthe probe described above (Fig. 1) showed protection ofthree fragments of about 72 to 74 nt (i.e., the size expectedfor transcripts from the normal hPBG-D cap site), whereasno such band was found with mouse reticulocyte RNA (Fig.2; the mouse PBG-D mRNA sequence is sufficiently differentnot to be detected with a human-specific probe). A minorprotected fragment corresponding to a transcript originatingfrom about -60 nt was also detected. In contrast, withpPBGhGH1+71 the level of hPBG-D cap site transcripts inMEL cells was reduced to below the background level ofsensitivity (Fig. 2).One explanation for the reduction in promoter activity in

pPBGhGH1+71 could be that the binding of NF-E1 or theCACCC factor is affected by the insertion of 71 bp ofpolylinker; however, this is not the case, as judged by invitro footprinting experiments (Fig. 3).To determine the spacing dependence more precisely, a

series of constructs containing different lengths of polylinkersequence were made as described in the legend to Fig. 1.

pPBG(-700)CAT

pPBG(-1 14)CAT

100-bp

Bcaccc nf-el

-114Clal

-114 91

-1 14 -91

+76pPBGhGH1

pPBGhGH1+71+76

caccc deletion mutant-87 +76

20 bpFIG. 1. Details of constructs containing hPBG-D promoter sequences. Fragments of the hPBGD erythroid promoter (a kind gift of M.

Goossens and P.-H. Romeo, Institut National de la Sante et de la Recherche Medicale, Creteil, France) (4) were cloned upstream of thepromoterless CAT or hGH genes. Symbols: anddO=, hPBG-D promoter and mRNA sequences, with nucleotide positions indicatedrelative to the cap site; , CAT gene (not to scale); M, hGH gene (not to scale and exons and introns not shown); -, polylinker sequencefrom pIC20H inserted at -91 nt in the hPBG-D promoter sequence. Positions of the CACCC motif and the NF-E1-binding site are indicated,as is the ClaI site in the polylinker sequence used for the BAL 31 digestion. pPBG(-700)CAT contains a 0.8-kilobase EcoRI-BamHI fragmentof the hPBG-D erythroid-specific promoter from about -700 to +76 bp inserted into the SmaI site upstream of the CAT gene in p22 (15),whereas in pPBG(-114)CAT hPBG-D sequences upstream of the PvuII site at -114 bp were removed from pPBG(-700)CAT. pPBGhGH1contains the hPBG-D sequence in pPBG(-114)CAT ligated into BamHI-HincII-cut pOGH (22) upstream of the hGH gene. pPBGhGH1+71contains the 71-bp Hindlll polylinker fragment from pIC20H (10) (modified to facilitate subsequent cloning steps by removal of the sequencebetween the BamHI and EcoRI sites) inserted into the BalI site at -91 bp between the NF-E1-binding site (CCTTATC) and the CACCCelement in the hPBG-D promoter contained in pPBGhGH1. Deletion mutants of pPBGhGH1+71 at the unique Clal site within the polylinkersequence were obtained by digestion with BAL 31 nuclease. The CACCC deletion mutant was formed by inserting the BalI-BamHI restrictionfragment between -87 and +76 bp of hPBG-D into HincII-BamHI-digested pOGH. To obtain site-directed mutagenesis of the GATAAG andCACCC motifs in the hPBG-D promoter (using the method of Kunkel [9]), a HindIII-BamHI fragment containing the hPBG-D sequence from-114 to +76 bp was subcloned into the phagemid pTZ18U (Bio-Rad catalog no. 170-3576). The oligonucleotides used for mutagenesis of theNF-E1-binding site were as follows: for the coding strand, 5'-AAGCTGATGGGCCTCATCTCTCTTTACCCACC-3' (containing a singlepoint mutation at -75 bp) and 5'-AAGCTGATGGGAATTCTCTCTITACCCACC-3' (containing three mutations at -78, -77, and -74 bp);and for the CACCC-binding site (coding strand), 5'-CTGCAGGCCCGGGCCTTCCTGTGGCC-3' (containing three mutations at -105, -104,and -103 bp).

7117111=11

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(A)- 87bp + 76bp

+±F 1 bp

v 0 - +*

(B)

194

1 2 3123

lnr!

118-

72-a-bp

FIG. 2. Effect of insertion of polylinker sequence between theNF-Ei-binding site and the CACCC motif on the hPBG-D promoteractivity in erythroid and nonerythroid cells. (A) Details of probeused for hybridization and Si protection analysis. Symbols: ---,polylinker sequences; and -, 87 bp of 5'-flanking sequenceand the first 76 bp of exon 1 of the hPBG-D gene. (B) Si-protectedfragments separated by electrophoresis, with HaeIII fragments of4.X174 run as size markers (positions shown on the left). Probe washybridized with 50 ,ug of mouse reticulocyte RNA (lane 1), MELcells transfected with pPBGhGH1 (lane 2), and MEL cells trans-fected with pPBGhGH+71 (lane 3). For measurement of RNAtranscripts, a 218-nt fragment of pPBGhGH1+71 containing se-quences between the Sall site in the inserted polylinker and theBamHI site at +76 bp in hPBG-D gene was selectively labeled at theBamHI end on the noncoding strand. Si nuclease protection wasperformed as described by Weaver and Weissmann (26). Hybridiza-tion was carried out by using probe plus 50 ,g of total RNA,followed by treatment with 20 U of Si nuclease (BoehringerMannheim Biochemicals) at 37°C for 60 min. After ethanol precip-itation, protected products were resolved by denaturing 6% poly-acrylamide gel electrophoresis.

Transfection of these constructs into MEL cells showed thatthe promoter activity was progressively reduced with inser-tions of up to about 25 bp between the NF-El-binding siteand the CACCC motif, when a residual promoter activity ofabout 20% of the wild-type value was reached (Fig. 4). Therewas no evidence for periodic changes in promoter activitywith respect to the number of helical turns ofDNA inserted.Work in other laboratories has shown that the CACCC

motif mediates other cell-specific signals such as the gluco-corticoid responsiveness of the tryptophan oxidase andother genes (20, 21). It is likely, therefore, that the CACCCmotif, and the protein that binds to it, is one example of aclass of general regulatory elements which mediate a number

+I11 bpW. T.

C O - +co

0

O

* wF

.I-

* 3

- w_ 0 _ _ .--

a

] CACCJ

1 IC ACCICACC

jEr

I.U'

~ m

-U'a _

_-

Ua :-e a. .4" m

,. mol --

W..

Ia ' _ ...

a

U

FIG. 3. Footprinting of the NF-El-binding site and the CACCCmotif in the wild-type hPBG-D promoter and promoters containing11- and 71-bp polylinker insertions. Plasmid DNAs from pPBGhGH1, pPBGhGH1+71, and pPBGhGH1+11 constructs were linear-ized at the BamHI site at +76 bp in the hGH gene and 5' end labeledwith [32P]ATP and T4 polynucleotide kinase; the promoter insertwas isolated after secondary digestion at the HindIII site in thevector upstream of the hPBG-D sequences extending to -114 bp.Nuclear protein preparation and footprint analysis were performedas described previously (15, 16). About 5 ng of end-labeled DNAwas incubated with 0 or 80 1±1 (40 p.g) of crude nuclear proteinextract from MEL cells in the presence of 3 jig of poly(dI-dC) poly(dI-dC) and in the presence (+) or absence (-) of 100 ngof double-stranded competitor oligonucleotide DNA (aG2; from theNF-Ei-binding site of the mouse a-globin gene [15]). After limitedDNase I digestion, DNA was purified and resolved on denaturing6% polyacrylamide gels. Footprints over the NF-Ei-binding site andthe CACCC motif are bracketed.

of cell-specific signals by interactions with specific transcrip-tion factors such as NF-Ei and the glucocorticoid receptor.However, unlike the situation for the NF-Ei and theCACCC factors in our experiments, optimal induction byglucocorticoids does depend on the precise spacing of theCACCC motif and the glucocorticoid response element, with

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0)

-6 80

60 1

C)

00

.-~~~~--~~~ m -(~~~H vectoraln0 10 20 30 40 50 60 70

Size of insert ( bp )FIG. 4. Effects of insertion of different lengths of polylinker sequence between the NF-E1-binding site and the CACCC motif on the

hPBG-D promoter activity. The levels of hGH produced after transient transfection of the different constructs into MEL cells are givenrelative to the level of the wild-type promoter (pPBGhGH1) ± standard errors. Each point is the average of two experiments, each involvingtwo to three separate transfections with every construct.

optimal induction at a periodicity of approximately 10 bpindicating a requirement for alignment on one face of theDNA helix (20, 21). A requirement for stereospecific align-ments has also been found for simian virus 40 early promoterelements (24) but not in several other systems, for example,in relation to interactions between the TATA motif and thebinding site for the GALA transcriptional activator in theyeast galactokinase gene promoter (19), the immunoglobulingene octamer transcriptional activator-binding site (27), andthe adenovirus major late transcription factor-binding site(2). It has already been shown that activity of the chicken,-globin enhancer can be affected by mutating sites inaddition to NF-E1 (18), and it will be interesting to learnwhether interactions occur between NF-E1 and other factorsthat bind in proximity to the promoter and enhancer ele-ments of various globin genes. A point of further interest inthe case of the hPBG-D promoter is the role of the NF-E2-binding site at -160 nt, which recent data have impli-cated in the large increase in PBG-D gene transcription thatoccurs during late induction of MEL cell differentiation (11,12), although in fact this site is deleted in the mouse PBG-Dpromoter (1). This finding implies either that an NF-E2 siteexists elsewhere in the mouse PBG-D gene or that its rolecan be replaced by that of another control region, possiblythe tandem duplication of the CACCC motif at about -100nt. Another difference between the human and mouse PBG-D promoters is that the transcription initiation point in themouse gene is about 40 nt downstream of the NF-E1-bindingsite (1), suggesting that in the mouse PBG-D gene this siteplays a role in positioning RNA polymerase for transcriptioninitiation similar to the role of the TATA-box factor.

This work was funded by the Cancer Research Campaign.

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Grandchamp. 1989. The mouse porphobilinogen deaminasegene. Structural organization, sequence and transcriptionalanalysis. J. Biol. Chem. 264:14829-14834.

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5. deBoer, E., M. Antoniou, V. Mignotte, L. Wall, and F. Grosveld.1988. The human P-globin promoter; nuclear protein factors anderythroid specific induction of transcription. EMBO J. 7:4203-4212.

6. Evans, T., M. Reitman, and G. Felsenfeld. 1988. An erythrocyte-specific DNA-binding factor recognizes a regulatory sequencecommon to all chicken globin genes. Proc. Natl. Acad. Sci.USA 85:5976-5980.

7. Galson, D. L., and D. E. Housman. 1988. Detection of twotissue-specific DNA-binding proteins with affinity for sites in themouse intervening sequence 2. Mol. Cell. Biol. 8:381-392.

8. Hall, C. V., P. E. Jacob, G. M. Ringold, and F. Lee. 1983.Expression and regulation of Escherichia coli lacZ gene fusionsin mammalian cells. J. Mol. Appl. Genet. 2:101-109.

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11. Mignotte, V., J. F. Elequet, N. Raich, and P.-H. Romeo. 1989.Cis- and trans-acting elements involved in the regulation of theerythroid promoter of the human porphobilinogen deaminasegene. Proc. Natl. Acad. Sci. USA 86:6548-6552.

12. Mignotte, V., L. Wall, E. de Boer, F. Grosveld, and P. H.Romeo. 1989. Two tissue-specific factors bind the erythroidpromoter of the human porphobilinogen deaminase gene. Nu-cleic Acids Res. 17:37-54.

13. O'Prey, J., J. Chester, B. J. Thiele, S. Janetzki, S. Prehn, J.Fleming, and P. R. Harrison. 1989. The promoter structure andcomplete sequence of the gene encoding the rabbit erythroidcell-specific 15-lipoxygenase. Gene 84:493-499.

14. Perkins, N. D., R. H. Nicolas, M. A. Plumb, and G. Goodwin.1989. The purification of an erythroid protein which binds toenhancer and promoter elements of haemoglobin genes. NucleicAcids Res. 17:1299-1314.

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cleod, G. Goodwin, and P. Harrison. 1989. GATAAG; a cis-control region binding an erythroid-specific nuclear factor witha role in globin and non-globin. Nucleic Acids Res. 17:73-92.

16. Plumb, M. A., and G. H. Goodwin. 1988. Detection of sequence-specific DNA-protein interactions by the DNA footprintingtechnique, p. 139-164. In J. M. Walker (ed.), Methods inmolecular biology, vol. 4. New nucleic acids techniques. Hu-mana Press, Clifton, N.J.

17. Plumb, M. A., V. V. Lobenkov, R. H. Nicolas, C. A. Wright, S.Zavou, and G. H. Goodwin. 1986. Characterisation of chickenerythroid nuclear proteins which bind to the nuclease hypersen-sitive regions upstream of the pA and P-globin genes. NucleicAcids Res. 14:7675-7693.

18. Reitman, M., and G. Felsenfeld. 1988. Mutational analysis of thechicken P-globin enhancer reveals two positive-acting domains.Proc. Natl. Acad. Sci. USA 85:6267-6271.

19. Ruden, D. M., J. Ma, and M. Ptashne. 1988. No strict alignmentis required between a transcriptional activator binding site andthe TATA box of a yeast gene. Proc. Natl. Acad. Sci. USA85:4262-4266.

20. Schule, R., M. Muller, C. Kaltschmidt, and R. Renkawitz. 1988.Many transcription factors interact synergistically with steroidreceptors. Science 242:1418-1420.

21. Schule, R., M. Muller, H. Otsuka-Murakami, and R. Renkawitz.1988. Cooperativity of the glucocorticoid receptor and the CA

CCC-box binding factors. Nature (London) 332:87-90.22. Selden, R., K. B. Howie, M. E. Rowe, H. M. Goodman, and

D. D. Moore. 1986. Human growth hormone as a reporter genein regulation studies employing transient gene expression. Mol.Cell. Biol. 6:3173-3179.

23. Superti-Furga, G., A. Barberis, G. Schaffner, and M. Busslinger.1988. The -117 mutation in Greek HPFH affects the binding ofthree nuclear factors to the CCAAT region of the gamma globingene. EMBO J. 7:3099-3107.

24. Takahashi, K., M. Vigneron, H. Matthes, A. Wildeman, M.Zenke, and P. Chambon. 1986. Requirement of stereospecificalignments for initiation from the simian virus 40 early pro-moter. Nature (London) 319:121-126.

25. Wall, L., E. de Boer, and F. Grosveld. 1988. The human ,-globingene 3' enhancer contains multiple binding sites for an eryth-roid-specific protein. Genes Dev. 2:1089-1100.

26. Weaver, R. F., and C. Weissmann. 1979. Mapping of RNA by amodification of the Berk-Sharp procedure: the 5' termini of 15SP-globin mRNA precursor and mature 10S ,-globin mRNA haveidentical map co-ordinates. Nucleic Acids Res. 7:1175-1193.

27. Wirth, T., L. Staudt, and D. Baltimore. 1987. An octameroligonucleotide upstream of a TATA motif is sufficient forlymphoid-specific promoter activity. Nature (London) 329:174-178.

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