Proc. Natl. Acad. Sci. USAVol. 93, pp. 3664-3668, April 1996Biochemistry

Transcription of the human corticotropin-releasing hormone genein NPLC cells is correlated with Z-DNA formation

(supercoiling/intron/Z-DNA antibody/laser crosslinks)

STEFAN WOLFL*t, CAMILO MARTINEZt, ALEXANDER RICH*§, AND JOSEPH A. MAJZOUB$§*Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; and tHarvard Medical School, Childrens Hospital, Boston, MA 02115

Contributed by Alexander Rich, December 7, 1995

ABSTRACT The intron of the corticotropin-releasinghormone (corticoliberin; CRH) gene contains a sequence ofover 100 bp of alternating purine/pyrimidine residues. Wehave used binding of a Z-DNA-specific antibody in metabol-ically active, permeabilized nuclei to study the formation ofZ-DNA in this sequence at various levels of transcription. Inthe NPLC human primary liver carcinoma cell line, activationof cAMP-dependent pathways increased the level of transcrip-tion, while adding glucocorticoids inhibited transcription ofthe CRH gene. These cells respond in a manner similar tohypothalamic cells. Z-DNA formation in this sequence wasdetected at the basal level of transcription, as well as afterstimulation with forskolin. Inhibition of transcription bydexamethasone abolished Z-DNA formation. Z-DNA forma-tion in the MYC gene (c-myc) was affected differently in thesame experiment. Thus, changes in Z-DNA formation in theCRH gene are gene specific and are linked to the transcriptionof the gene.

Physiological processes in chromatin are associated withchanges in the torsional strain of DNA that in turn facilitateconformational changes. Under negative superhelical stress,some sequences readily undergo a conversion from the righthanded B-DNA to the left handed Z-DNA conformation.Though the dynamics of Z-DNA formation have been studiedin many systems, our knowledge of Z-DNA formation inendogenous DNA sequences in vivo is still very limited.Recently a method was developed that allows us to study theformation of Z-DNA in individual genes (1, 2). Based on theprotocol of Jackson and Cook (3) cells are encapsulated inagarose microbeads and metabolically active, permeabilizednuclei are prepared. These nuclei retain their functions ofreplication and transcription (4). Biotin-labeled Z-DNA-specific monoclonal antibodies are allowed to diffuse intothese nuclei. The amount of antibody used is in a range ofconcentration that does not alter the B- to Z-DNA equilib-rium. Thus, it is below the level that induces Z-DNA formationbut high enough to saturate by binding naturally occurringZ-DNA sites (5). Irradiation with a 10-ns pulse of UV-laserlight at 266 nm crosslinks the antibody to the bound sequenceelements. Upon restriction digestion, antibody-bound frag-ments are affinity purified by binding to streptavidin. Follow-ing proteolysis, the restriction fragments that were in theZ-DNA conformation in the cell nucleus are available foranalysis.

Corticotropin-releasing hormone (corticoliberin; CRH) is a41-amino acid peptide (6) which plays a central role inmodulating the hypothalamic-pituitary-adrenal (HPA) axis inresponse to stress or homeostatic maintenance (7). SecretedCRH stimulates the production of corticotropin (ACTH) inthe anterior pituitary gland, which in turn causes the produc-tion of glucocorticoids in the adrenal cortex. Synthesis and

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

3664

secretion of CRH in hypothalamic cultures and cell lines arestimulated by ligands which activate either the cAMP-dependent protein kinase A (PKA) or the protein kinase C(PKC) pathway (8). Glucocorticoids mediate the physiologicalresponses to stress and inhibit CRH release from the hypo-thalamus through a negative-feedback loop. The stimulationand inhibition ofCRH expression by PKA and glucocorticoids,respectively, are mediated by changes in CRH gene transcrip-tion (9, 10).

Analysis of the human CRH gene revealed a sequence ofalternating purine and pyrimidine residues more than 100 bplong in the intron of the CRH gene. Stretches of alternatingpurine and pyrimidine residues are known to predispose asequence to adopt the left handed Z-DNA conformation (11).Negative supercoiling is required to stabilize the left handedZ-DNA conformation. Although most of the DNA in the cellnucleus is negatively supercoiled, this negative supercoiling ismasked by higher order chromatin structures, such as nucleo-somes. However, transcription and replication continuouslyinfluence the torsional stress of the DNA molecule, leading tolocal changes in negative supercoiling. For example, as RNApolymerase moves along its template it leaves behind nega-tively supercoiled DNA and creates positively supercoiledDNA in front as it pushes through the double helix (12).

Besides the potential ability to form Z-DNA, this part of theCRH gene also attracted our attention because it may beinvolved in inhibition by glucocorticoids, though it does notcontain an obvious glucocorticoid-receptor binding site. Intransient-transfection experiments in NPLC cells with a lucif-erase reporter gene driven by the CRH sequences -914 to+127, glucocorticoid administration did not inhibit basalactivity and it reduced PKA-stimulated expression by onlyone-fourth (9). However, this expression construct does notcontain the Z-DNA-forming region of the CRH gene, raisingthe possibility that this or other omitted regions of the CRHgene may account for the potent glucocorticoid inhibition seenin vivo. To investigate whether Z-DNA formation could playa role in the transcriptional regulation of the gene, we used themethod described above and studied the formation of Z-DNAwhen transcriptional was enhanced by the PKA pathway orinhibited by glucocorticoids.

MATERIALS AND METHODSGrowth of NPLC Cells. NPLC/PRF/5, a human hepatocel-

lular carcinoma-derived cell line containing the hepatitis Bvirus genome, obtained from Knowles (13), was grown in anatmosphere containing 10% CO2 in Dulbecco's modifiedEagle's medium (DMEM) (GIBCO) supplemented with 10%

Abbreviations: CRH, corticotropin-releasing hormone; PKA, cAMP-dependent protein kinase A; PKC, protein kinase C; PMA, phorbol12-myristate 13-acetate.tPresent address: Hans-Knoll-Institut fur Naturstoff-Forschung,07745 Jena, Germany.§To whom reprint requests should be addressed.

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(vol/vol) fetal bovine serum, 50 units of penicillin (GIBCO)per ml, and 50 ,Ag of streptomycin per ml (GIBCO). Cells wereplated 48 h prior to experiments and were subconfluent whencollected.mRNA Isolation and Northern Blotting. NPLC cells were

treated with 10 ,tM forskolin (Sigma)/0.1 ,uM dexamethasone(Sigma)/0.1 ,M phorbol 12-myristate 13-acetate (PMA; Sig-ma), or the ethanol vehicle alone for 6 h before beingharvested. mRNA was isolated by using standard RNA isola-tion techniques by collecting cells in a guanidinium thiocya-nate solution (4 M guanidinium thiocyanate/2 M cesiumchloride/0.5% sodium N-lauroylsarcosine/25 mM sodium ci-trate/1 mM 2-mercaptoethanol), underlaid with 5.7 M cesiumchloride, and spun at 65,000 rpm in a 100.2 ultracentrifugerotor (Beckman) for 18 h. The RNA pellet was dissolved in 0.3M sodium acetate and collected by precipitation in ethanol.RNA was resuspended in diethyl pyrocarbonate (DEPC)-treated H20, and the total yield was measured by using UVspectroscopy. A total of 10 ,Ag of RNA was run on a 1.4%agarose gel containing formaldehyde and then transferred toa nylon membrane. RNA was immobilized by UV crosslinking(Stratalinker, Statagene; 254 nm; 120 mJ) and hybridized witha 32P-labeled CRH cRNA probe. Membranes were exposed tox-ray film or quantified by using a Phosphorlmager (MolecularDynamics) (14).Treatment with Forskolin and Dexamethasone. Solutions

containing 10 ttM forskolin (Sigma), 0.1 ,tM dexamethasone(Sigma), or forskolin and dexamethasone dissolved in ethanoland added to 108 cells. The same amount of vehicle was addedto the otherwise untreated control cells. After 1 h of incuba-tion, cells were collected and washed twice in phosphate-buffered saline (0.2 g of KCI per liter/0.2 g of KH2PO4 perliter/8 g of NaCl per liter/1.15 g of Na2HPO4 per liter).

Preparation of Agarose-Encapsulated, Metabolically ActiveNuclei. Agarose-encapsulated, metabolically active nucleiwere prepared as described (1, 3, 5). Permeabilization of theencapsulated nuclei was carried out in buffer A (130 mMKCl/1 mM MgCl2/1 mM Na2ATP/10 mM Na2HPO4, adjustedto pH 7.4 if necessary with KH2PO4) containing 0.25% TritonX-100 on ice for 20 min. The detergent was removed bywashing the beads five times in buffer A without Triton X-100.

Transcription, Replication, and Antibody Binding. A vol-ume of 300 ,ul of agarose encapsulated nuclei (containing -2X 106 cells) was used in the antibody-binding assay. Highconcentrations of all four rNTPs [final concentration (f.c.) 6mM] and of all four NTPs (f.c. 2 mM) were added, and thevolume was adjusted to 500 ,Al. A total of 2.4 ,tg of Z-DNA-specific, biotinylated monoclonal antibody Z22 (15) was addedto each sample. The samples were left at room temperature for2 h to allow for antibody binding.

Crosslinking of Antibody. Crosslinking was done with a10-ns pulse of the fourth harmonic wavelength at 266 nm of aNd-YAG laser. The pulse energy was about 12 mJ with thebeam focused on an area of 100 mm2. The samples wereexposed in a UV-grade quartz cuvette with 5-mm pathlength.

Isolation of the Z-DNA Fragments. After crosslinking, allsamples were treated as described (1). In short, unboundproteins and antibody were removed by high salt treatment (f.c.>2 M NaCl). Genomic DNA was digested with the restrictionenzyme Alu I. The supernatant of the Alu I digest was addedto streptavidin-coated magnetic particles. Biotinylated anti-bodies crosslinked to the Z-DNA fragments were allowed tobind for 30 min. Magnetic particles were then washed inten-sively to remove nonspecifically bound restriction fragments.Z-DNA-specific fragments were released from the magneticparticles by proteinase K digestion. Residual protein wasremoved by extraction with phenol and subsequent precipita-tion in ethanol.

Detection of Specific Z-DNA Fragments by PCR. PCRprimers used to amplify specific Alu I restriction fragments of

the CRH gene were as follows: CRH163U(1142), ATGTGCG-CCGCGGAG; CRH163L(1304), TCTTAAGGAATAGTCC-GCGAAC; CRH352U(368), AAGATGGTGGGACTC; andCRH352L(719), CAACAGATATTTATCGCC. The numbergives the length of the PCR product; primer corresponds toeither the upper strand (U) or lower strand (L). The numberin parentheses corresponds to the nt position of the primerrelative to the 5' end of the human CRH gene sequence.PCR was carried out in 50 gl of 1.5 mM MgCl2/0.1% Triton

X-100/70mM Tris-HCl, pH 8.8, containing 1 ,tl of the Z-DNAfraction (50 ,tl total) and 30 pmol of each primer. After a 5-mindenaturation at 98°C, samples were kept at 85°C, and 2 ptl ofa start mixture containing dNTPs (5 mM) and 2.5 units of TaqDNA polymerase (Ampli-Taq, Perkin-Elmer) was added tothe hot sample. A total of 35 cycles of amplification werecarried out as follows: 94°C for 1 min and 65°C for 45 sec. Todetect the three MYC Z-DNA fragments, we used the PCRprimers listed previously (1). PCR was done as described abovewith 35 cycles of 94°C for 1 min and 62°C for 45 sec.

Southern Transfer and Hybridization. The gels containingthe PCR products for the CRH gene were washed in alkalinesolution for denaturation, and the amplified DNAs weretransferred to a nylon membrane by capillary transfer. DNAwas immobilized byUV crosslinking (Stratalinker; 254 nm; 120mJ) and hybridized with a 32P-labeled CRH cRNA probe.

RESULTSTranscription of the CRH Gene in NPLC Cells. Human

primary liver carcinoma cells (NPLC) have been shown toexpress the endogenous CRH gene (16). The transcriptionalactivity of the gene can be influenced by glucocorticoids andphorbol esters. As in hypothalamic cultures (17), agents thatactivate either the PKA or the PKC pathway stimulate tran-scription, while glucocorticoids inhibit transcription of thegene.To ensure that the specific stock of NPLC cells used in the

following experiments exhibit similar regulation of the CRHgene by the PKA and PKC pathways, we measured thetranscription of CRH in response to forskolin and dexameth-asone. Forskolin stimulates the cAMP pathway via adenylatecyclase, leading to an activation of genes that contain acAMP-responsive element (CRE). The CRH gene containsone CRE in its promoter area which has been shown tomediate regulation by the PKA pathway (9, 18, 19). Dexa-methasone is a potent synthetic glucocorticoid which inhibitsCRH expression (9, 20). Cells were grown in DMEM mediumand the solvent (ethanol); dexamethasone; the phorbol esterPMA, an activator of the PKC pathway; forskolin; or bothdexamethasone and either PMA or forskolin were added.After overnight incubation, cells were collected and mRNAwas prepared. Fig. 1 demonstrates that dexamethasone re-duced expression of endogenous CRH by more than a factor ofeight. PMA administration enhanced expression about 2-fold.Forskolin stimulated mRNA expression approximately 4.5-fold, which is more than previously seen (16). This may be dueto the slightly different characteristics of the stock of NPLCcells used in these experiments. Dexamethasone abolished thestimulation by either PMA or forskolin. NPLC cells treated foronly 6 h also exhibited similar regulation (data not shown).These results indicate that the regulation of the CRH gene inthe NPLC cells used is regulated similar to its regulation inhypothalamic cell cultures.Z-DNA Is Formed in the CRH Gene in Metabolically Active

Nuclei. Forskolin, forskolin and dexamethasone, or dexameth-asone alone was added to approximately 108 cells for 1 h. Cellswere then collected and Z-DNA-forming sequence elementswere isolated according to published protocols (1, 2). Afterencapsulation in agarose microbeads, cellular membraneswere lysed, and the nuclear envelope was permeabilized by

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A

5

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z3-

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163 bp ->

B

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M 1 2 34M

1234

1234

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FIG. 1. Northern blot analysis of the CRH mRNA in NPLC cells.(Upper) Autoradiograph showing the specific CRH mRNA bands.(Lower) Display of the quantitation obtained by using a Phosphorlm-ager. Cells were treated with ethanol (Control), dexamethasone (0.1,lM DEX), 0.1 ,tM PMA, both dexamethasone and PMA, forskolin(10 ,M FSK), or both forskolin and dexamethasone (FSK/DEX). TheCRH mRNA level of control cells was arbitrarily set at 1. Other valueswere calculated relatively. Error bars are shown.

using Triton X-100. Biotin-labeled, Z-DNA-specific monoclo-nal antibodies were diffused into these agarose-encapsulated,metabolically active permeabilized cell nuclei, and NTPs were

added to reactivate transcription and replication. The concen-tration of antibody used does not significantly alter the B to Zequilibrium (1, 5). Antibody-bound DNA fragments werecrosslinked to the biotinylated Z-DNA-specific antibodies witha 10-ns pulse of 266-nm laser light. Unbound proteins wereremoved by high-salt washes, the DNA was digested with AluI, and Alu I restriction fragments diffused out of the agarosemicrobeads. Streptavidin-coated magnetic particles were

added to the supernatant containing the restriction fragmentsof the genomic DNA, capturing the fragments that were

covalently linked to biotinylated Z-DNA-specific antibodies.Unbound restriction fragments were removed by serial washes.Z-DNA-specific restriction fragments were then released byproteinase K digestion and further purified by extraction withphenol and precipitation with ethanol.

Fig. 2 shows the results ofPCR carried out with a primer pairspecific for the Alu I restriction fragment of the CRH gene thatcontains the alternating purine/pyrimidine stretch. Ethidiumbromide staining of an agarose gel (Fig. 2A) shows theCRH-specific PCR product (arrow) together with a variety ofbackground bands generated by the primer pair. The productis present during basal transcription (Fig. 2A, lane 1) and afterforskolin treatment (Fig. 2A, lane 2). No specific band is seenafter treatment with both forskolin and dexamethasone (Fig.2A, lane 3) or dexamethasone alone (Fig. 2A, lane 4). Toensure that only one band corresponds to the specific Alu Irestriction fragment of the CRH gene, the PCR products weretransferred to a nylon membrane and hybridized with a

CRH-specific probe. Fig. 2B shows a short exposure and Fig.2C a long exposure of the same blot hybridized with a

radioactively labeled CRH-specific probe. The band represent-ing the Z-DNA-forming CRH restriction fragment is present inuntreated cells (Fig. 2B, lane 1), is somewhat stronger afterforskolin treatment (Fig. 2B, lane 2), and is still detectable butweaker when both forskolin and dexamethasone were added tothe cells (Fig. 2B, lane 3). Even after long exposure this band

FIG. 2. Products of PCR when using the primer pair specific for theAlu I restriction fragment of the human CRH gene containing aZ-DNA-forming sequence element. Z-DNA-specific-antibody-boundAlu I restriction fragments from NPLC cells were analyzed. Lane M,marker; 1-h incubation with ethanol (control/solvent), lane 1; fors-kolin (10 j/M), lane 2; both forskolin (10 /aM) and dexamethasone (0.1/lM) lane 3; dexamethasone (0.1 ,M), lane 4. (A) Ethidium bromide-stained agarose gel: the position of the 163-bp CRH-specific PCRfragment is indicated. (B and C) Autoradiographs after hybridizationwith a CRH-specific probe. Only the above indicated 163-bp-longfragment is visible. Short exposure (B) shows bands in lanes 1 and 2;long exposure (C) shows bands in lanes 1, 2, and 3.

is not found when the cells were treated by dexamethasonealone (Fig. 2C, lane 4). Control reactions (not shown) using aPCR primer pair for another Alu I restriction fragment of theCRH gene did not yield any product in the Z-DNA fraction butgave a strong response when unfractionated genomic DNA wasused.Z-DNA in theMYC Gene. To determine whether the changes

in the Z-DNA level were specific for the CRH gene or moregeneral resulting from the treatment of the cells, the effect offorskolin and glucocorticoid on the Z-DNA-forming elementsof the human MYC gene was examined. Previously, three AluI restriction fragments of the human MYC gene (myc.Z1,myc.Z2, and myc.Z3) was shown to form Z-DNA in U-937 cellswhen the gene is actively transcribed (1). We used identicalPCR primer pairs to ask if the restriction fragments thatformed Z-DNA in U-937 cells could be found in the Z-DNA-specific restriction fragments of NPLC cells as well. Fig. 3shows the results of PCR experiments for myc.Z1, myc.Z2, andmyc.Z3. The primer pair for myc.Z1 gives rise to a barelyvisible PCR product in the Z-DNA fraction of the cells treatedwith both forskolin and dexamethasone (Fig. 3, lane 3). Primerpairs for myc.Z2 and myc.Z3 give rise to PCR products in allfour Z-DNA fractions. The signal for myc.Z2 in the cellsincubated with both forskolin and dexamethasone (Fig. 3, lane3) seems to be somewhat stronger. These results for the MYCgene clearly show that intensity variations of the bands rep-resenting the Z-DNA-specific PCR products of the CRH geneare not due to general variations in the yield of restrictionfragments containing Z-DNA-forming elements. The strong-est signal for Z-DNA formation in the CRH gene is found incells treated with forskolin alone, followed by the signalobtained for untreated cells. In contrast, the signal for Z-DNAformation in the MYC restriction fragments Zl and Z2 isstrongest when NPLC cells are treated with both forskolin anddexamethasone. The signal for the MYC restriction fragmentZ3 is strongest in the two samples with forskolin. The aboveexperiments demonstrate that the formation of Z-DNA in theCRH gene correlates very well with the transcriptional activity

366 Biochemistry: Wiilf I et al.

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myc.Z1

256 bp ->

myc.Z2

301 bp ->

1 2 3 4 Mmyc8bp.

85 bp ->-

FIG. 3. PCR with primer pairs specific for the Alu I restrictionfragments of the human MYC gene containing Z-DNA-formingsequence elements and the same Z-DNA-specific fraction of Alu Irestriction fragments from NPLC cells as in Fig. 2. Lane M, marker;other lanes have 1-h incubation with ethanol (control/solvent), lane 1;forskolin (10 JLM), lane 2; both forskolin (10 ,tM) and dexamethasone(0.1 JLM), lane 3; or dexamethasone (0.1 ,jM), lane 4. Primer pairspecific for myc.Z1 gives rise to a 256-bp fragment; primer pair specificfor myc.Z2 gives rise to a 301-bp fragment, and primer pair specific formyc.Z3 gives rise to an 85-bp fragment.

of the gene. In contrast, the MYC gene is regulated in adifferent fashion, indicating that the changes in the Z-DNAlevel of the CRH gene are CRH specific.

DISCUSSIONA long stretch of an alternating purine/pyrimidine sequence isfound in the intron of the human CRH gene (Fig. 4A). Ourstudies using the binding of a Z-DNA-specific antibody inmetabolically active, permeabilized nuclei revealed that this

A 183 bp AluI restriction fragmentof the CRH geneCTGCCGCGAT GTGCGCCGCG GAGCCGGCTG CCCCTGCCtg

10 20 30 40

tgtacgtgtg tatgcatgta tgtgcgtgca tgtgtgtgt8

tgtatgcatgtgttatgcg tatgtgttgg tgtgtat?gc

cgcgtgcgtl cgtgtgtgt tgcgcgc?tg tTCGCGGACT160

ATTCCTTAAG ATTACAATAG TAG170 180

CRH{--E Ik\\"\I

Exon 1 Exon 2

183

IfI / IIZ

z

MYCPo P1 P2 P3

rF rr-> rr '

275 336 245i ii

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ZI Z2

sequence is found in the Z-DNA conformation in the tran-scribed gene. Z-DNA formation is detected at the basal levelof transcription. Stimulation of transcription by forskolinappears to increase the relative level of Z-DNA formation inthis DNA element. Downregulation of gene transcriptionstrongly reduces the level of Z-DNA formation. When tran-scription is further reduced, this restriction fragment is nolonger detected in the Z-DNA-specific preparation of Alu Irestriction fragments. These observations are consistent withprevious observations for the human MYC gene (1) andindicate that transcriptionally generated negative supercoilingplays a role in stabilizing Z-DNA in these nuclei. As describedby Liu and Wang (12), the degree of negative supercoiling willbe increased in the wake of the moving polymerase if transcrip-tion increases. After the polymerase has passed a Z-DNA-forming element, it will be stabilized in the Z-DNA conformationby transcriptionally generated supercoiling energy until negativesupercoils are released by the action of topoisomerases.What common features of these elements are found in the

Z-DNA conformation in transcribed genes in vivo? As men-tioned, all contain stretches of alternating purines/pyrimidines, predominantly GC or GT/AC. That predisposesa sequence for Z-DNA formation by lowering the energyrequired. The sequence of the human CRH gene is particularlysurprising since the alternating purine/pyrimidine repeat is113 bp long. Since most of the.sequences are (GT/AC)nrepeats, it has some obvious similarities to other (GT/AC)sequence elements which are found in some microsatellitesequences.

Cross-species comparison revealed that Z-DNA-formingelements are present at the same position in the rat, murine,and sheep CRH genes (21, 22). The length of the respectivefragments varies significantly between species. The Z-DNAelement in the human gene is 113 bp long, and the alternationof purines and pyrimidines is only interrupted by phase shifts.The rat and mouse genes have 30-bp-long potential Z-DNAelements with an alternating purine/pyrimidine sequence in-cluding some AT bases. In the sheep gene, the potentialZ-DNA-forming element is 53 bp long and consists largely of

FIG. 4. (A) Sequence of the CRHAlu I restriction fragment showing theZ-DNA motif in lowercase letters. (B)Position of the Z-DNA fragments inthe human CRH gene and in the hu-man MYC gene. Both genes are dis-played with exons (boxes; hatchedboxes indicate the translated section)and introns. Major transcription startsites are indicated by arrows. Alu Irestriction sites are marked by verticallines below. Restriction fragmentscontaining Z-DNA forming elementsare indicated by heavy horizontal bars.The number above indicates the lengthof the restriction fragment in basepairs. The numbers below at the flank-ingAlu I sites refer to the 5' end of thedisplayed fragment. CRH: 3.2 kbshown; transcription start, nt 916; in-tron, nt 1086-1885. MYC: 8 kb shown;transcription start PI: nt 2328; intron 1:nt 2882-4505; intron 2: nt 5278-6653.

---I -- 11 11 I1 --I I II

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a (GT/AC)n sequence. The presence of a Z-DNA motif in allsequenced mammalian CRH genes in a noncoding part makesit reasonable to consider that Z-DNA may contribute to theregulation of the CRH gene.Another question is the relationship between the position of

Z-DNA-forming elements and their role in gene expression.Fig. 4B shows representations of the human CRH and thehuman MYC gene. The single Z-DNA-forming element of theCRH gene is found in the intron, fairly close to the transcrip-tion start site. The Z-DNA-forming elements of the MYC geneare upstream of the gene and in the first intron. One of theseelements, Z2, is also close to the major transcription start siteof the gene. All positions make a stabilization of Z-DNA bytranscriptionally generated negative supercoiling possible. Apassing polymerase induces Z-DNA formation. It has beenshown that RNA polymerase cannot transcribe through Z-DNA (23). The subsequent polymerase is then blocked by theZ-DNA until that element is flipped back to the B-DNAconformation due to the action of topoisomerases. The loca-tion in the first intron may thus help to maintain a spacingbetween adjacent polymerases at high transcription levels. Thiscould be important for the proper processing of the transcript.The position of the Z-DNA-forming element close to the

transcription start site might influence the positioning oftranscription factors and the transcription apparatus at thetranscription start site. Enhanced affinity of topoisomerase IIand other proteins to DNA in the Z-DNA conformation (24,25), which masks binding sites for B-DNA-binding proteins, aswell as providing an energetic sink for negative supercoilsmight be needed to maintain relevant structures in the chro-matin of the gene. However, Z-DNA formation is probably nota primary mechanism for the activation of transcription, eventhough it may have other important functions.Could this topological transformation contribute to the

downregulation of the CRH gene by dexamethasone? Whenthe effect of dexamethasone on the transcription of theendogenous CRH gene is compared with its effect on thetransient expression of a CRH-promoter-luciferase-reportergene construct, more effective inhibition of the endogenousgene is observed (9). Further, basal expression is only sensitiveto dexamethasone inhibition in the endogenous gene. Twodifferences should be considered. Obviously, the expressionconstruct does not contain the complete CRH gene, but it hasa major part of the promoter that is sufficent for cAMP-induced expression. The expression construct is not integratedinto the genome, and, thus, its chromatin will not be repre-sentative for the gene. The effect observed in the transientexpression may thus represent only one part of the in vivosituation. Transient expression shows the effect mediated bythe direct interaction between the activated cAMP-dependenttranscription factor CREB and the activated glucocorticoidreceptor. This effect seems to be the major mode of glucocor-ticoid-mediated repression and is consistent with observationsmade for glucocorticoid repression of NF-KB- and AP-1-mediated transcription (26-28). Work on the mouse mammarytumor virus promoter shows that chromatin structure plays arole in the activating potential of the cAMP pathway (29) andthat glucocorticoids may alter the chromatin structure (30). Inthat study, glucocorticoid treatment induced a more activechromatin structure. In repression by glucocorticoids, how-ever, the presence of a Z-DNA-forming element may facilitate

the transition from the more active to the less active chromatinstructure. Although it has been demonstrated that Z-DNA isformed locally in a transcribed, unintegrated plasmid vector invitro (31), it may be able to contribute significantly to chro-matin organization only in an integrated gene.

This research was supported by grants from the National Institutesof Health, National Science Foundation, Office of Naval Research,and the American Cancer Society.

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