Proc. Nadl. Acad. Sci. USAVol. 84, pp. 2387-2390, April 1987Genetics

S1 nuclease analysis of a-globin gene expression in preleukemicpatients with acquired hemoglobin H disease after transfer tomouse erythroleukemia cells

(trans-acting factor/preleukemia/acquired hemoglobin disease)

J. HELDER AND A. DEISSEROTH*University of California, San Francisco Hematology/Oncology Unit, Veterans Administration Medical Center, San Francisco, CA 94121

Communicated by Helen M. Ranney, December 12, 1986 (receivedfor review June 26, 1986)

ABSTRACT The loss of a-globin gene transcriptionalactivity rarely occurs as an acquired abnormality during theevolution of myeloproliferative disease or preleukemia. To testwhether the mutation responsible for the loss of a-globin geneexpression (hemoglobin H disease) in these patients is linkedwith the a-globin genes on chromosome 16, we transferredchromosome 16 from preleukemic patients with acquiredhemoglobin H disease to mouse erythroleukemia cells andmeasured the transcriptional activity of the human a-globingenes. After transfer to mouse erythroleukemia cells, theexpression of human a-globin genes from the peripheral bloodor marrow cells of preleukemic patients with acquired hemo-globin H disease was similar to that of human a-globin genestransferred to mouse er'ythroleukemia cells from normal do-nors. These data showed that factor(s) in the mouseerythroleukemia cell can genetically complement the a-globingene defect in these preleukemia patients with acquired hemo-globin H disease and suggest that altered expression of a genein trans to the a-globin gene may be responsible for theacquisition of hemoglobin H disease in these patients.

Hemoglobin H disease is usually a familial hypochromicmicrocytic anemia associated with the presence of P-globintetramers (hemoglobin H) in the erythrocytes of the periph-eral blood or marrow. The excess of 13-globin chains in theerythrocytes of these patients is due to the loss of expressionof at least three of the four a-globin genes normally presentin diploid erythroid cells (1). Previous studies in our labora-tory (2), as well as those of Weatherall and coworkers (3, 4),have suggested that hemoglobin H disease can also beacquired during the evolution of preleukemia or myelopro-liferative disease. In these latter patients, the transcriptionalactivity of all four ofthe a-globin genes appears to be reducedto 10% of normal. Extensive structural analysis of thesetranscriptionally inert a-globin genes has failed to identifydifferences between the a-globin genes of these preleukemicpatients and those of normal individuals. Since the transcrip-tional activity of all four of the a-globin genes appears to bereduced to an equivalent degree, many workers have sug-gested that altered expression of a single gene, which affectsthe expression of all four a-globin genes on two separatehomologous chromosomes, is responsible for this disorderand is located in trans to the a-globin genes.To test whether the loss of transcriptional activity of all

four a-globin genes in these patients is due to a mutation thatis linked to the a-globin genes on chromosome 16, or islocated on another chromosome, we transferred the chro-mosome that carries the a-globin genes, chromosome 16 (5),from the peripheral blood or marrow cells of these patientsinto mouse erythroleukemia cells, and then we isolated

mouse erythroleukemia cell clones that contained humanchromosome 16 as the only human chromosome. We com-pared the expression of human a-globin genes from thehemoglobin H disease patients with the expression of a-globin genes from normal individuals after transfer ofeach tomouse erythroleukemia cells. Previous experiments hadshown that normal levels of human a- but not 4- (embryonic)globin gene expression have been observed when chromo-some 16 from normal donors is transferred from peripheralblood or marrow cells (nonerythroid as well as erythroid) intothe mouse erythroleukemia cells (6-9). Therefore, persis-tence ofthe low transcriptional activity ofthe human a-globingenes from the hemoglobin H disease patients after transferto mouse erythroleukemia cells would suggest that thepresence of a defect linked to each human a-globin gene onchromosome 16 is responsible for this disease. On the otherhand, equal levels of human a-globin gene expression in themouse erythroleukemia cells that contain human a-globingenes from normal or hemoglobin H disease patients wouldsuggest that the defect responsible for the disordered a-globingene expression is in trans to these genes.

SI nuclease analysis showed that the level of humana-globin mRNA in nine mouse erythroleukemia cell lines,which contained a-globin genes from four different acquiredhemoglobin H donors, was similar to that observed in fivemouse erythroleukemia cell lines with a-globin genes fromthree different normal donors. Restriction fragment poly-morphisms (10, 11) were used to document that both of thehomologues of human chromosome 16 from one of thepatients contained a-globin genes that displayed similarpatterns of expression after transfer to the mouse erythro-leukemia cells. These results suggest that the loss of a-globingene expression in these patients is due to a defect that islocated in trans to the a-globin genes.

METHODSHuman peripheral blood mononuclear cells (108 cells), iso-lated by Ficoll/Hypaque gradient centrifugation (12), werefused in the presence of polyethylene glycol with 107 mouseerythroleukemia cells using published methods (6). Each ofthe mouse erythroleukemia cells contained a full complementof mouse chromosomes. The absence of adenine phosphori-bosyltransferase activity in the mouse erythroleukemia cellspermitted us to select for the retention ofhuman chromosome16 in the hybrid cells. Metaphase spreads of the hybrid cellswere generated by described methods (5). Analysis of a-globin genes in patients and hybrid cells was performed bySouthern blot analysis as reported (9, 10, 13). S1 nucleaseanalysis was performed on 20 ,ug of total RNA extracted (14)

*To whom reprint requests should be addressed at: Hematology/Oncology Section (111H), Veterans Administration Medical Cen-ter, 4150 Clement Street, San Francisco, CA 94121.

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

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2388 Genetics: Helder and Deisseroth

from hybrid mouse erythroleukemia cells exposed for 72 hrto dimethylformamide by the method of Orkin and Goff (15).Fragments of the 0.55-kilobase (kb) HindIII fragment of theantisense strand ofhuman a-globin cDNA clone JW101 (a giftof B. Forget) and the 0.4-kb BstEII/Sst I fragment of theantisense strand of the mouse a-globin gene (a gift of P.Leder) were used as probes.

RESULTS

Properties ofHybrid Cells. As shown in Table 1, we isolatedfive independently derived hybrid mouse erythroleukemiaclones with human a-globin genes from three different normaldonors, six hybrid mouse clones with human a-globin genesfrom the peripheral blood of three different acquired hemo-globin H disease donors, and three hybrid mouse clones withhuman a-globin genes from the marrow cells of an additionalacquired hemoglobin H disease donor. We were also able touse restriction fragment length polymorphisms to isolatehybrid cell clones that contained the a-globin genes fromeach of the two homologues of human chromosome 16present in the cells of one of the preleukemic donors (patient2) as shown in Table 1 and in Fig. 1.As shown in Table 1 and Fig. 1, EcoRI/BamHI digests and

Southern blots were used to identify the presence of each ofthe two homologues of chromosome 16 from patient 2, whichcarries either a 13- or a 16-kb EcoRI/BamHI a-globin generestriction fragment in mouse erythroleukemia cell lines619B3, 622A1, 632A1, and 638B16. These polymorphicfragments reflect the presence oftwo or three human a-globingenes on chromosome 16. This variant, which has also beenreported among normal individuals (10, 11), is also present inrelatives of patient 2, who are normal with respect to a-globingene expression, and is not thought to be related to thepresence of defective a-globin gene expression in patient 2.

S1 Nuclease Analysis of Human a,- and a2-Globin GeneExpression in Mouse Erythroleukemia Cells. As shown in Fig.2 (lanes 1 and 2), when mixtures of human and mouse32P-end-labeled a-globin DNA probes were mixed with eitherhuman or mouse reticulocyte RNA, the S1 nuclease assaywas totally specific for mouse and human globin mRNA,

1 2 3 4 5 6 1 9 10 11 12 13

FIG. 1. Southern analysis ofEcoRI/BamHI restriction fragmentpolymorphisms of the human a-globin genes in hybrid mouseerythroleukemia cells that contain human chromosome 16 frompreleukemic donors (patients 2 and 4). The identity of the hybrid celllines from which digested DNA was added in each lane is as follows:lane 1, 707-5; lanes 2 and 3, blank; lane 4, 707-1; lane 5, 707-2; lane6, X HindIII marker; lane 7, 638B16; lane 8, 632A1; lane 9, 622A1;lane 10, 619B3. Lanes 11 and 12, blank; lane 13, mouse DNA. Thefilter was hybridized with a 32P-labeled Hinfi 0.9-kb fragment ofJW101 (human a-globin cDNA).

permitting simultaneous assessment of human and mousea-globin mRNA sequences for each hybrid cell. Trial RNAtitration experiments with each probe and the mixtures wereconducted to ensure that the hybridizations were run in probeexcess, thereby validating the measurement of human andmouse RNA in each sample (lanes 3-5). All hybridizations ina given experiment were developed on the same autoradi-ograph. Therefore, the ratio of human/mouse a-globinmRNA could be used to compare levels of human a-globinmRNA among the different hybrid clones. This ratio isindependent ofthe variability in human a-globin gene expres-sion that would arise from (i) differences in the degree towhich each hybrid cell responds to the dimethylformamideused to stimulate erythroid gene expression, (it) differencesin the quantitative recoveries that may occur during RNAisolation and ethanol precipitation, (iii) the absolute amountofRNA added to each hybridization mixture (as long as probeexcess is maintained), (iv) the specific activities ofthe probesused, and (v) variability in exposure of the autoradiograph.As explained in Fig. 2, two different lengths of fragments,

Table 1. Human a-globin gene expression in mouse erythroleukemia hybrid cells

Ratio humanDonor a-Globin* a-globin mRNA/

Donor cell gene restriction mouse a-globin mRNA MeanCell line cell type source fragment marker Exp. 1 Exp. 2 Exp. 3 Exp. 1 Exp. 2 Exp. 3

620D1 Normal 1 PB Not done 1.92 3.33 7.91617D6 Normal 2 PB Not done 3.35 13.3 8.41599-5 Normal 3 PB Not done 4.89 7.78 9.99617E4 Normal 2 PB Not done 3.65 9.69 8.16 3.26 7.50 7.94617D1 Normal 2 PB Not done 2.49 3.41 5.26271-8 Patient 1 PB Not done 2.92 13.9 9.14619B3 Patient 2 PB 13-kb EcoRI/BamHI 3.97 14.8 8.84622A1 Patient 2 PB 16-kb EcoRI/BamHI 3.14 7.76 8.86632A1 Patient 2 PB 16-kb EcoRI/BamHI 3.97 8.94 2.62638B16 Patient 2 PB 16-kb EcoRI/BamHI 3.32 11.9 10.8 3.80 8.25 7.45683A2 Patient 3 PB 14-kb Hpa I 4.74 7.57 6.4707-1 (sample 1) Patient 4 Marrow 13-kb EcoRI/BamHI 4.37 7.54 7.22707-1 (sample 2) Patient 4 Marrow 13-kb EcoRI/BamHI 5.32 7.50707-2 (sample 1) Patient 4 Marrow 13-kb EcoRI/BamHI 5.9 8.49707-2 (sample 2) Patient 4 Marrow 13-kb EcoRI/BamHI 4.50 5.93 4.09 5.23 7.01707-5 (sample 1) Patient 4 Marrow 13-kb EcoRI/BamHI 2.00 3.67 6.21707-5 (sample 2) Patient 4 Marrow 13-kb EcoRI/BamHI 4.92

Seventy to 100% of all metaphase spreads contained human chromosome 16. PB, peripheral blood. Probes used for experiments 1, 2, and3 were made in independent nick-translations and are therefore of different specific activities, giving different ratios of human a-globin/mousea-globin mRNA from experiment to experiment. However, the ratios within each experiment can be compared.*Southern analysis of restriction fragment polymorphism shown in Fig. 1.

Proc. Natl. Acad. Sci. USA 84 (1987)

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Proc. Natl. Acad. Sci. USA 84 (1987) 2389

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FIG. 2. SI nuclease analysis of total RNA from hybrid mouse erythroleukemia cells that contain a-globin genes from normal donors andfrom preleukemic donors affected with acquired hemoglobin H disease. JW101 human a1-globin cDNA probe (1000 cpm) (0.55-kb HindIIIantisense strand 32P-labeled at the HindIII site in the coding block by the Klenow reaction) and mouse a-globin gene probe (1000 cpm)(BstEII/Sac I antisense strand 32P-end-labeled at the BstEII site on exon III) were added to each reaction mixture. RNA was added to the reactionmixture in each lane as follows: lane 1, 50 ng of human reticulocyte globin mRNA; lane 2, 50 ng of mouse reticulocyte globin mRNA; lanes3-7, total RNA from each of the following hybrid mouse cells, respectively, 617D1 (15 ,kg), 617D1 (45 ,ug), 617D1 (75 ,ug), 683A2 (20 /Lg), and632A1 (20 jug). Lane 8, 32P-end-labeled 4X174 Hae III DNA markers. Lanes 9-14, 20 ,ug of total RNA from hybrids 271-8, 617D6, 707-1, 707-2,707-5, and 622A1, respectively. Lane 15, blank. Lane 16, 20 ,ug from hybrid cell 620D1; lanes 17 and 18, 4X174 Hae III DNA markers. Variationin the intensity of the autoradiographs in lanes 9-12 may be due to differences in inducibility of the hybrid cells and/or recovery after ethanolprecipitation, since the intensity of the mouse and human bands vary in a similar manner. Lanes 19-22, 20 ,ug of RNA of hybrid cells 638B16,619B3, 599-5, and 617E4, respectively. All hybridization reaction mixtures were incubated at 38°C for 3 hr and treated with 1000 units of SigmaSi nuclease and subsequently run on 5% urea acrylamide gels as described (9, 15). The human a1-globin cDNA fragments that are protectedby human a-globin mouse globin mRNA are 268 and 172 nucleotides long. The 268-nucleotide fragment is protected by human a2-globin mRNA,while the shorter 172-nucleotide human a1-globin cDNA fragment is protected by human a2-globin mRNA. The shorter length is due to thepresence of nucleotide sequence differences between a1- and a2-globin mRNA, which are recognized by Si nuclease 172 nucleotides from theend-labeled HindIlI site in the probe (15). The mouse DNA fragment protected by mouse globin mRNA is 200 nucleotides long.

172 and 268 nucleotides long, of the human a1-globin cDNAprobe are protected by human a-globin mRNA, due to thepresence of sequence differences between human a1-globinmRNA and human a2-globin mRNA. This sequence differ-ence results in S1 nuclease digestion of a site in the humana1-globin cDNA probe, which is 172 nucleotides from the32P-end-labeled HindIII site (15) of the probe, when it isprotected by human a2-globin mRNA.As shown by the data presented in Fig. 2 and Table 1, the

ratio of human/mouse a-globin mRNA was the same forhybrid mouse erythroleukemia cells that contained humanchromosome 16 from normal versus preleukemic donors withacquired hemoglobin H disease. In addition, the hybrid cellsthat were shown by restriction fragment length polymor-phism analysis to contain different human a-globin genealleles from the same patient (patient 2) were shown topossess similar amounts of human a-globin mRNA as shownin Fig. 2. Finally, hybrid mouse erythroleukemia cells, whichcontained human a-globin genes from the marrow of apreleukemic patient (patient 4) with acquired hemoglobin Hdisease, exhibited levels of human a-globin mRNA indistin-guishable from those found in hybrids that contained humanglobin genes from normal donors. The marrow cells used forcell fusion were taken from this patient at a time whenerythroid and megakaryocytoid hematopoietic cells in themarrow were dysplastic morphologically, and 90% of thehematopoietic cells had been replaced by the preleukemicclone as judged by the a-/p-globin mRNA ratio of 0.1 in theerythroid marrow cells.

DISCUSSIONThe results reported in this paper show that transfer of thechromosomes bearing the a-globin genes from the peripheralblood or marrow cells of preleukemia patients with acquiredhemoglobin H disease to mouse erythroleukemia cells resultsin levels of the human a-globin mRNA equal to those found

when a-globin genes from normal donors are transferred tothe mouse erythroleukemia cells.Our previous studies ofpatients with hemoglobin H disease

acquired in the setting of preleukemia or myeloproliferativedisease (2) had shown that the transcription of all foura-globin genes had been reduced to 0.1 ofnormal and that theratio of a1-/a2-globin gene expression was similar to thatfound in normal erythroid cells. These data (2) had suggestedthat the hematopoietic cells of these patients are either ahomogeneous leukemic population, in which the erythroidcells contain four a-globin genes, all of which are transcrip-tionally active at a level that is 10% that found in normal cells,or that the hematopoietic population present in these patientswas composed of 90% leukemic cells, in which all foura-globin genes were totally inactive, and of 10% normalresidual nonleukemic hematopoietic cells, in which the a-globin genes were fully active.The a-/,8-globin chain and mRNA ratios in the marrow and

peripheral blood of these patients were below 0.1 at the timeat which the chromosomal transfer experiments were per-formed, suggesting that at least 90% of the erythroid cellswere abnormal in these patients (4). Ifthe a-/,B-globin mRNAratios of 0.1 had been due to the presence of a mixturecomposed of 10% normal cells, in which all of the a-globingenes were totally transcriptionally active, and 90%dysplastic cells, which contained a-globin genes totallyinactivated by mutations in cis, one would have expected themajority of the mouse erythroleukemia cell lines to exhibittotal absence of human globin gene expression and a few toexhibit totally normal levels of a-globin transcriptional ac-tivity. None of the mouse erythroleukemia cell lines witha-globin genes from these patients was devoid of humana-globin gene expression, and all showed a level of humana-globin gene expression indistinguishable from that presentin normal mouse erythroleukemia cell lines with a-globingenes from normal donors. This suggests that the defect thatcaused decreased a-globin gene expression is in trans to thea-globin genes.

Genetics: Helder and Deisseroth

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2390 Genetics: Helder and Deisseroth

It is also possible, however, that the results of ourexperiments are due to the presence of long-lived normallymphocytes in the peripheral blood cells of patients used forcell fusion, which were not derived from the preleukemicclone. To test this possibility, we fused the marrow cells ofone of the preleukemic patients (patient 4) with mouseerythroleukemia cells and isolated human chromosome 16-containing clones. All of the hybrid mouse clones gavehuman a-globin mRNA levels equal to those found in hybridclones with a-globin genes from normal individuals. In thepopulation of marrow cells from patient 4, which were usedfor this fusion, dysplastic cells were present in the erythroidand megakaryocytoid series, and the a-/,3-globin mRNAratio was 0.1. Using the degree to which the marrowa-/f-globin ratio has been lowered (0.1) as an index for thedegree to which the preleukemic clone has replaced thenormal marrow hematopoietic cells of the patient, the prob-ability that all three of the hybrid clones (707-1, 707-2, and707-5) contained human a-globin genes from normal cells is1/1000. Moreover, since 20% of the peripheral blood mono-nuclear cells could have been normal nonleukemic lympho-cytes, and 10% of the rest of the mononuclear cells werenonleukemic (on the basis of the a-/P-globin mRNA ratio of0.1 in the peripheral blood), the probability that normal cellscontributed to all six of the hybrid clones derived from theperipheral blood of the preleukemic patients was (0.3)6 or 7X 10-4. Thus, the overall probability that all of the hybridmouse erythroleukemia cells containing human a-globingenes from marrow and peripheral blood were derived fromnonleukemic cells remaining in the patients was 7 x 10-7, anexceedingly low value.

Thus, our results suggest that the low transcriptionalactivity of all of the a-globin genes present in the erythroidcells of these patients before transfer is due to a mutation thatis asyntenic to these genes and that the transcriptional defectin these leukemic cells can be complemented by a trans-acting factor present in the mouse erythroleukemia cells.Formal proof of a trans defect in this disease will ultimately

rest on the identification of the specific trans mutation or theisolation of a marker for the leukemic cell line in thesepatients.

The authors are indebted to Drs. David Weatherall and DouglassHiggs for their suggestions and discussions as well as their donationof the peripheral blood from three of their patients with acquiredhemoglobin H disease and leukemia. This work was supported bygrants to A.D. from the National Institutes of Health (R01 HL29300),the Veterans Administration Merit Review, and the Council forTobacco Research, U.S.A., Inc.

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489-500.8. Charnay, P., Triesman, R., Mellon, P., Chao, M., Axel, R. &

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Glaister, D., Marks, B., Wang, A., Colbert, D. & Deisseroth,A. (1985) Blood 65, 707-711.

10. Higgs, D., Old, J., Pressley, L., Clegg, J. & Weatherall, D.(1980) Nature (London) 284, 632-635.

11. Goossens, M., Dozy, A., Embury, S., Zachariades, Z.,Hadjiminos, M., Stamatoyannopoulos, G. & Kan, Y. W.(1980) Proc. Natl. Acad. Sci. USA 77, 518-521.

12. Boyem, A. (1968) J. Clin. Lab. Invest. Suppl. 97, 21, 77-89.13. Southern, E. (1975) J. Mol. Biol. 98, 503-517.14. Favaloro, J., Treisman, R. & Kamen, R. (1980) Methods

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