Nucleic Acids Research, Vol. 18, No. 23 7093
Developmental regulation of the human zeta globin in
gene
transgenic mice
E.A.Spangler, K.A.Andrews and E.M.Rubin* Divisions of Cell and Molecular Biology and Research Medicine, University of California, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA Received June 22, 1990; Revised and Accepted November 9, 1990
ABSTRACT We have characterized the expression of the human zeta (r) gene, which encodes an embryonic a-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site 11 (HSII) from the distal 5* region of the human globin gene cluster potentiates expression of the v globin gene. In the absence of the HSII fragment, no r expression is observed. Expression of the human t gene in mice parallels expression of a murine embryonic a-like globin gene (x). Thus, expression of the human ¢ gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in globin gene expression can be analyzed in detail. a
a
globin gene regulation. Minor hypersensitive sites are located close to each of the genes of the cluster, and their presence or absence correlates with the expression of those genes (4,5). Major hypersensitive sites map upstream and downstream of the , globin cluster, and are present in erythroid tissues throughout development (5,6). When constructs containing only the minor hypersensitive sites together with either a human fetal or adult globin gene are introduced into mice, the genes are developmentally regulated, but the level of expression is variable (7,8,9,10,11,12). The addition of DNA fragments containing some or all of the major hypersensitive sites allows -y and globin transgenes to be expressed consistantly at levels approaching those of endogenous globin genes (13,14). This suggests that sequences proximal to the genes are essential for the developmental switch in globin gene expression, while the role of distal sequences, including the major hypersensitive sites, is to potentiate high level expression of those genes. Distal DNA sequences normally involved in the regulation of a globin gene expression have not been reported, and expression of human globin in transgenic mice has been achieved only when the ca gene is coupled to DNA fragments spanning some or all of the major hypersensitive sites from the human globin cluster (15,16,19). We describe here the expression in transgemc mice of the human v gene, which encodes an embryonic a-like globin. Moderate levels of adult human a and globin gene expression can be obtained in mice when a small fragment encompassing major hypersensitive site II (HSII) alone is associated with the gene (17,18,19). We demonstrate that this HSII fragment is also sufficient to potentiate expression of the human embryonic a-like globin (0) in mice. In addition, we find that the human embryonic gene follows the same developmental pattern of regulation as the murine embryonic oa-like globin gene a
INTRODUCTION The genes of the human a globin and globin loci are expressed in a specific temporal pattern, and in specific hemopoietic tissues during development. The embryonic globins (¢ and e) are expressed in the yolk sac blood islands until about the fifth week of gestation. At that time, adult globin (a) and fetal , globin (A,y and G^y) genes begin to be expressed in the liver. The liver is gradually replaced as the major site of hemopoiesis by the spleen and bone marrow, which express the adult globin genes (a, 6 and () (1). A similar developmental pattern of globin gene expression exists in mice. Hemopoiesis begins in the yolk sac at about day eight of gestation, and the fetal liver becomes the major site of hemopoiesis at about day ten. There is no fetal globin in mice, thus the mouse fetal liver produces only adult hemoglobins (2,3). Although the mechanisms that regulate developmental changes in globin gene expression are not completely understood, several lines of evidence indicate that two categories of DNase I hypersensitive sites (major and minor) are important in human a
*
To whom correspondence should be addressed
(x).
METHODS Transgenic Mice Transgenic mice were generated essentially as described by Hogan et al. (20). Embryos were obtained by breeding C57BL/6 x SJL F 1 hybrid parents. DNA was microinjected into
7094 Nucleic Acids Research, Vol. 18, No. 23 the pronuclei of one-cell embryos at a concentration of 2.0 nglpl. Injected embryos were cultured overnight in M 16 medium (20), and those that divided to two cells were transferred to the oviduct of a pseudopregnant female and allowed to develop to term. Transgenic animals were identified by Southern blot analysis of tail DNA, using a 4.8 kb genomic EcoRI fragment that spans the entire v gene as a probe. The probe fragment was labelled with 32p to a specific activity of 1I x 109 cpm/,ug using the random primer method (21).
Preparation of DNA for Microinjection An 882 bp fragment spanning HSII (17) was provided by Peter Curtin (University of California, San Francisco), and was inserted into the BamHI site of vector pUC 19. A 4.8 kb EcoRI fragment containing the v gene was isolated from cosmid pCL9, which contains the entire human a globin cluster (22), and was ligated into an EcoRI site located 21 bp from the HSII fragment. The 5' to 3' orientation of the r gene relative to the HSII fragment was the same as is found in the genome for the genes of the: globin cluster. For injection into mouse embryos, a 5.7 kb PvuI-HindIII fragment containing 777 bp of the original HSII insert linked to the r gene was isolated by preparative agarose gel electrophoresis. The fragment was recovered by electroelution, followed by concentration on an Elutip column (Schliecher & Schuell, Inc.) and ethanol precipitation. The DNA pellet was resuspended in injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) and filtered (0.2 Arm) prior to microinjection.
Preparation of Mouse RNA and DNA RNA was prepared as described by Chirgwin et al. (23). DNA was prepared by digestion of tissue with proteinase K, followed by chloroform extraction and ethanol precipitation. To obtain fetal and embryonic tissue samples, transgenic males were bred with normal females. The day that the copulation plug was observed was designated as day 0. Embryonic yolk sacs were collected at day 9 and fetal livers were collected at day 16. In each case, 10 or more tissue samples were pooled in the final RNA preparation. In some experiments RNA was prepared at day 16 from individual fetal livers, and DNA was prepared from the remainder of each fetus. Adult transgenic mice were treated with phenylhydrazine (25 ng/gm body weight injected intraperitoneally daily for 4 days) to induce production of nucleated red blood cells, and RNA was prepared from 200 1l of peripheral blood
units of M-MLV Reverse Transcriptase (Bethesda Research Laboratories). Reactions were incubated at 37°C for 1 hour. Nucleic acids were precipitated with ethanol, resuspended in denaturing sample buffer (95 % formamide) and analyzed on 8 % acrylamide/7M urea gels.
RESULTS In order to study the regulation of the human zeta gene during development, we established lines of transgenic mice and examined expression in staged embryos. The construct used to generate those mice is shown in figure 1. It consisted of a 4.8 kb DNA fragment containing the human v globin gene, linked to a 777 bp fragment spanning erythroid-specific hypersensitive site II (HSII). Three founder transgenic animals were identified by Southern blot hybridization, and were designated Z 1, Z5 and Zl1. To characterize the inheritance of the transgene, each of the founder animals was bred and offspring were screened by Southern blot hybridization. A typical result obtained using a human v probe is shown in figure 2. In DNA from the founder 0
10
30
20
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50
40
60
70
80
90 VI
Cy Ay VP 1~~~~~~~~~~~~~~~~~~~~~1 I' 6
C
t030 kb
p
;
Human p Locus
\
Human X Locus I,
HSII-Zeta Construct 0
1
2
3
4
5
6 kb
Figure 1. HSII-¢ construct. The human ce and /3 globin loci are shown, with the approximate positions of the genes represented by black boxes. The locations of the major DNaseI hypersensitive sites are indicated by arrows, and are identified by roman numerals. DNA fragments used to build the HSII-t construct are indicated by dotted lines, and features of the construct are represented as follows: HSII fragment, black line; ¢ exons, black boxes; ¢ introns, open boxes; ¢ 5' and 3' flanking sequences, hatched boxes.
-
on the fifth day.
Primer Extension Assay Oligonucleotide primers were 5'-end-labelled with 32p using T4 Kinase (Bethesda Research Laboratories), and were separated from unincorporated label using Nuctrap columns (Stratagene). The sequences of the primers were as follows: human ¢, 5'-TCTTGGTCAGAGACATGGCG-3'; mouse x, 5'-TGAGGGTTGGAGGCTGCGCT-3'; mouse x, 5'-CAGGCAGCCTTGATGTTGCTT-3'. Approximately 10,000 cpm of labelled primer was added to 10 Ag RNA. The RNA/primer mixture was evaporated to dryness and resuspended in 7.5 AI of 0.2M NaCl. Samples were sealed in glass microcapillaries, heated to 80°C for 2 minutes, and then incubated at 30°C for 1 hour. After hybridization, each sample was added to a tube containing a reverse transcription reaction mixture. The final reaction volume was 50 /d, and contained 50 mM Tris, pH 8.3, 30 mM NaCl, 75 mM KCl, 10 mM DTT, 3 mM MgCl2, 1 mM each dGTP, dATP, dCTP and TTP, 20 units of RNasin (Promega) and 200
.i.,
w
s
a.i
Figure 2. Southern blot analysis of mice from the Z 11 transgenic line. DNA from the Z 11 founder (lane 1) and 5 offspring (lanes 2-6) was digested with EcoRI, blotted and probed with a 4.8 kb genomic DNA fragment spanning the human ¢ globin gene. Transgenic animals are characterized by a band at 4.8 kb that hybridizes to the human probe. Additional bands visible in lane I are the result of partial digestion of the DNA. Lanes 7 and 8 contain an amount of plasmid DNA equivalent to I and 2 copies of human ¢ per haploid genome respectively.
. wE
Nucleic Acids Research, Vol. 18, No. 23 7095
(ZI 1, lane 1), and in DNA from each of the transgenic offspring (lanes 2,4,5), there is a 4.8 kb fragment that hybridizes to the r probe. By comparing the intensity of the transgenic r signal to the intensity of the signal in lanes 7 and 8 which contain known amounts of r plasmid DNA, we estimate that the transgene copy number in the Z 11 line is -2-4 per haploid genome. Similarly, four of the eight offspring of Z5 were found to be transgenic, and the transgene copy number was estimated to be 1-2 per haploid genome. No transgenic offspring of Z1 were identified, and we presume that this animal is a mosaic. To verify that the entire HSII- construct was present in the Z5 and Z 11 lines, duplicate blots were probed for HSII. A band of the expected size was detected in each transgenic lane (data not shown). On the basis of these results, the Z5 and Z 11 transgenic lines were both chosen for further analysis. A primer extension assay was used to determine if the human v globin gene was expressed in the two transgenic lines, and at what stages of development. Total RNA was isolated from the yolk sacs of day 9 embryos and from the peripheral blood of adult transgenic animals. The embryonic tissues were derived from matings between transgenic males and normal females, and RNA was prepared from pools of 10 or more tissue samples to insure an adequate supply of material. Embryonic RNA samples were primed with a 1:1 mixture of the mouse x and human r primers. Adult RNA samples were primed independently with the mouse a primer and the mixed embryonic primers (x and O), because the ca and r extension products migrate at overlapping positions on the gel. The results of this experiment are shown in figure 3. In both transgenic lines, correctly initiated human r and mouse x transcripts are present in embryonic erythroid cells (lane 1). In adult erythroid cells, mouse a globin transcripts are present (lane 3), but human v and mouse x transcripts are undetectable (lane 2). The relative intensities of the primer extension bands approximate the relative amount of each transcript in the RNA sample. By assuming that 50% of the pooled tissue from which the embryonic RNA samples were prepared was transgenic, we estimate that human r transcripts are present at greater than 60% the level of mouse x transcripts in embryonic erythroid tissue of the Z5 line, and at greater than 30% the level of mouse x transcripts in embryonic tissue of the Z 11 line. The level of Z5
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human r and mouse x transcripts in transgenic adult RNA preparations is below the limit of detection of our assay. From this we conclude that transcription of the human r transgene is reduced to less than 1 % the level of the mouse a globin gene in adult animals. The mouse x globin gene is not expressed in the definitive erythroid cells of the fetal liver. To establish if there is a parallel reduction in the number of human r transcripts per animal at the same developmental stage, transgenic males were mated with normal females and total RNA from individual day 16 fetal livers was analyzed. DNA was isolated from each fetus, and transgenics were identified by Southern blot hybridization. Representative transgenic and non-transgenic RNA samples were assayed for the presence of transcripts from the mouse ae, the mouse x and the human r globin genes by primer extension. The results of this experiment are shown in figure 4. In both transgenic and control samples there is a strong mouse ax signal, and no detectable signal for mouse x or human t. Thus, transcription of the human r transgene has ceased in parallel with the switch from expression of murine embryonic to adult globin genes.
2 3
-
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-
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Mnx
M xj_ .. __
Mxx
Figure 3. Primer extension analysis of RNA from embryonic and adult erythroid tissues. In the panel on the left, RNA samples are from the Z5 line. In the panel on the right, RNA samples are from the Z 11 line. Lane 1 contains RNA prepared from embryonic yolk sacs, primed with a 1: 1 mixture of the human r and mouse x primers. Lane 2 contains adult RNA primed with a 1:1 mixture of the human ¢ and mouse x primers, and lane 3 contains adult RNA primed with the mouse ca primer.
primer
Mx
_
-
.-_ -L
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---
---J
Ms
Figure 4. Primer extension analysis of RNA prepared from individual fetal livers. In panel A, RNA samples are from the Z5 line. In panel B, RNA samples are from the Z1 1 line. The number above each lane identifies the RNA preparation used in that primer extension reaction. Transgenic samples are designated by a T and non-transgenic controls are designated by a C. The primer(s) used in each reaction are indicated below the lanes. Lane d9C contains RNA prepared from yolk sacs of transgenic embryos at day 9, primed with a 1:1 mixture of the mouse x and human r primers. Lane C contains the products of extension by all three primers with no added template RNA. Lane M contains a DNA size marker.
7096 Nucleic Acids Research, Vol. 18, No. 23
DISCUSSION We have constructed transgenic mice that harbor the human embryonic ¢ globin gene associated with a 777 bp DNA fragment that spans erythroid-specific DNase I hypersensitive site II from the distal 5' region of the globin gene cluster, and have examined the expression of the human gene during development. In six lines of transgenic mice that contained the same ¢ globin DNA fragment without HSII, no expression of the human gene was detected at any stage of development (E. R., unpublished). This result mirrors what is observed when human oa globin is introduced into mice without an enhancer (15,16). Using the HSII-D construct, we have observed correctly initiated transcription of the human gene in the erythroid cells of the embryonic yolk sac. We conclude that the embryonic gene, like the adult globin genes, requires the presence of an erythroidspecific enhancer for expression in transgenic mice. In addition, we find that the timing of expression of the human ¢ gene in erythroid tissues parallels that of the mouse x gene. From this we infer that like the murine embryonic globins, the human embryonic globin transgene is expressed only in the primitive erythroid cells of the yolk sac. The results of this study suggest that the 4.8 kb fragment containing the zeta globin gene with 0.55 kb of 5' and 2.75 kb of 3' flanking sequence contains all of the information necessary to specify that the gene be expressed in primitive erythroid cells and not be expressed in definitive erythroid cells. The evolutionary conservation of an approximately 300 bp region located immediately 5' to both the human r and the mouse x genes suggests that this noncoding sequence may have an important regulatory function (24). It is unlikely that expression of the human ¢ gene in murine erythroid cells is limited to a particular time in development as a direct result of its association with the HSII fragment. When the HSII fragment is linked to an adult human globin gene (a or () in transgenic mice, the adult gene can be expressed in adult tissues (17,18,19). Moreover, a human globin transgene in an HSLI- construct is not developmentally regulated; the adult gene is expressed in both primitive and definitive murine erythroid cells (E.S, K.A. and E.R., unpublished). Developmental regulation of the human r globin gene when linked to one of the major hypersensitive sites does not require the presence of other globin genes in cis. A similar observation was reported by Raich et al. (25), who found that developmental regulation of the human embryonic globin gene (e) is correct when that gene is linked to four major hypersensitive sites. In contrast, the loss of developmental regulation of human 'y and globin gene expression when the individual genes are introduced into mice linked to the major hypersensitive sites of the globin cluster is well documented (26,27,28). It has been suggested that the switch from fetal to adult globin gene expression involves a competition between the individual globin genes for interaction with the major hypersensitive sites (27,28). The outcome of this competition is thought to be influenced by positive and/or negative factors whose presence is developmentally regulated. According to this model, in the absence of competition the major hypersensitive sites form a stable interaction with a single gene and drive expression of that gene at all developmental stages (27,28). This model cannot account for the observed developmental regulation of embryonic globin transgenes, thus it seems likely that the mechanism for a
silencing expression of embryonic globin
genes at
later
stages
of development is fundamentally different from that which regulates the transition from fetal to adult gene expression.
Further gene transfer experiments will allow clarification of the role of proximal regulatory sequences and of their interactions with the major hypersensitive sites in the developmental regulation of globin gene expression. The mechanism of a globin gene switching during development has been refractory to experimental studies because of the lack of a cell culture or transgenic mouse system in which those genes were appropriately regulated. The use of constructs including major hypersensitive sites from the human j globin cluster has made it possible to obtain tissue-specific a globin expression (15,16), and our results suggest that this is a system in which the developmental regulation of expression of the a-like globin genes can also be studied. Because the murine pattern of embryonic a globin expression mirrors what occurs in humans, the transgenic mouse should provide an accurate and informative model system in which to study human a globin gene switching. After the submission of this manuscript Higgs et al. (29) reported the identification of sequences located far upstream of the human a globin genes that activate high level a globin expression both in MEL cells and in transgenic mice. The use of constructs containing those sequences, in place of the activating sequence from the 3 globin cluster, may provide additional insights into the requirements for a globin switching.
ACKNOWLEDGEMENTS We thank Shirley Clift and Deepa Kumar for identifying the zeta transgenic founders, and David Asarnow and Linda Couto for critical reading of the manuscript. This work was supported by NIH-HL20985 (EMR) and NRSA-HL07279 (EAS). This research was conducted at the Lawrence Berkeley Laboratory (Department of Energy Contract DE-AC03-76SF00098 to the University of California).
REFERENCES 1. Stamatoyannopoulos, G. & Nienhuis, A.W. (1987) The Molecular Basis of Blood Diseases. eds. Stamatoyannopoulos, G., Nienhuis, A.W., Leder, P. & Majerus, P.W. (Saunders, Philadelphia, PA). 2. Craig, M.L. & Russell, E.S. (1964) Developmental Biology 10, 191 -201. 3. Fantoni, A., Bank, A. & Marks, P.A. (1967) Science 157, 1327- 1328. 4. Groudine, M., Kohwi-Shigematsu, T., Gelinas, R., Stamatoyannopoulos, G. & Papayannopoulou, T. (1983) Proc. Natl. Acad. Sci. 80, 7551 -7555. 5. Tuan, D., Solomon, W., Li, Q. & London, I.M. (1985) Proc. Natl. Acad. Sci. 82, 6384-6388. 6. Forrester, W.C., Takegawa, S., Papayannopoulou, T., Stamatoyannopoulos, G. & Groudine, M. (1987) Nuc. Acids Res. 15, 10159-10177. 7. Chada, K., Magram, J., Raphael, K., Radice, G., Lacy, E. & Costantini, F. (1985) Nature 314, 377-380. 8. Magram, J., Chada, K. & Costantini, F. (1985) Nature 315, 338-340. 9. Townes, T.M., Lingrel, J.B., Chen, H.Y., Brinster, R.L. & Palmiter, R.D. (1985) EMBO J. 4, 1715-1723. 10. Chada, K., Magram, J. & Costantini,F. (1986) Nature 319, 685-689. 11. Kollias, G., Wrighton, N., Hurst, J. & Grosveld, F. (1986) Cell 46, 89-94. 12. Rubin, E.M., Ronghua, L., Cooper, S., Mohandas, N. & Kan, Y.W. (1988) Am. J. Hum. Genet. 42, 585-591. 13. Grosveld, F., Blom van Assendelft, G., Greaves, D.R. & Kollias, G. (1987) Cell 51, 975-985. 14. Talbot, D., Collis, P., Antoniou, M., Vidal, M., Grosveld, F. & Greaves, D.R. (1989) Nature, 338, 352-355. 15. Ryan, T.M., Behringer, R.R., Townes, T.M., Palmiter, R.D. & Brinster, R.L. (1989) Proc. Natl. Acad. Sci. 86, 37-41. 16. Hanscombe, O., Vidal, M., Kaeda, J., Luzzatto, L., Greaves, D.R. & Grosveld, F. (1989) Genes & Dev. 3, 1572-1581. 17. Curtin, P.T., Liu, D., Liu, W., Chang, J.C. & Kan, Y.W. (1989) Proc. Natl. Acad. Sci. 86, 7082-7086. 18. Ryan, T.M., Behringer, R.R., Martin, N.C., Townes, T.M., Palmiter, R.D. & Brinster, R.L. (1989) Genes & Dev. 3, 314-323.
Nucleic Acids Research, Vol. 18, No. 23 7097 19. Rubin, E.M., Spangler, E.A., Curtin, P., Witowska, E., Clift, S. & Lubin, B. (1989) Blood, 74, 823a. 20. Hogan, B., Costantini, F. & Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.) 21. Feinberg, A.P. & Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 22. Lau, Y.-F. & Kan, Y.W. (1983) Proc. Natl. Acad. Sci. 80, 5225-5229. 23. Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. & Rutter, W.J. (1979) Biochem. 18, 5294-5299. 24. Leder, A., Weir, L. & Leder, P. (1985) Mol, & Cell. Biol. 5, 1025- 1033. 25. Raich, N., Ebens, A.J., Papayannopoulou, T., Enver, T. & Stamatoyannopoulos, G. (1989) Blood 74, suppl. 1, 7a. 26. Enver, T., Ebens, A.J., Forrester, W.C. & Stamatoyannopoulos, G. (1989) Proc. Natl. Acad. Sci. 86, 7033-7037. 27. Enver, T., Raich, N., Ebens, A.J., Papayannopoulou, T., Costantini, F. & Stamatoyannopoulos, G. (1990) Nature 344, 309 -313. 28. Behringer, R.R., Ryan, T.M., Palmiter, R.D., Brinster, R.L. & Townes, T.M. (1990) Genes & Dev. 4, 380-389. 29. Higgs, D.R., Wood, W.G., Jarman, A.P., Sharpe, J., Lida, J., Pretorius, I.-M. & Ayyub, H. (1990) Genes & Dev. 4, 1588-1601.
Developmental regulation of the human zeta globin in
gene
transgenic mice
E.A.Spangler, K.A.Andrews and E.M.Rubin* Divisions of Cell and Molecular Biology and Research Medicine, University of California, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA Received June 22, 1990; Revised and Accepted November 9, 1990
ABSTRACT We have characterized the expression of the human zeta (r) gene, which encodes an embryonic a-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site 11 (HSII) from the distal 5* region of the human globin gene cluster potentiates expression of the v globin gene. In the absence of the HSII fragment, no r expression is observed. Expression of the human t gene in mice parallels expression of a murine embryonic a-like globin gene (x). Thus, expression of the human ¢ gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in globin gene expression can be analyzed in detail. a
a
globin gene regulation. Minor hypersensitive sites are located close to each of the genes of the cluster, and their presence or absence correlates with the expression of those genes (4,5). Major hypersensitive sites map upstream and downstream of the , globin cluster, and are present in erythroid tissues throughout development (5,6). When constructs containing only the minor hypersensitive sites together with either a human fetal or adult globin gene are introduced into mice, the genes are developmentally regulated, but the level of expression is variable (7,8,9,10,11,12). The addition of DNA fragments containing some or all of the major hypersensitive sites allows -y and globin transgenes to be expressed consistantly at levels approaching those of endogenous globin genes (13,14). This suggests that sequences proximal to the genes are essential for the developmental switch in globin gene expression, while the role of distal sequences, including the major hypersensitive sites, is to potentiate high level expression of those genes. Distal DNA sequences normally involved in the regulation of a globin gene expression have not been reported, and expression of human globin in transgenic mice has been achieved only when the ca gene is coupled to DNA fragments spanning some or all of the major hypersensitive sites from the human globin cluster (15,16,19). We describe here the expression in transgemc mice of the human v gene, which encodes an embryonic a-like globin. Moderate levels of adult human a and globin gene expression can be obtained in mice when a small fragment encompassing major hypersensitive site II (HSII) alone is associated with the gene (17,18,19). We demonstrate that this HSII fragment is also sufficient to potentiate expression of the human embryonic a-like globin (0) in mice. In addition, we find that the human embryonic gene follows the same developmental pattern of regulation as the murine embryonic oa-like globin gene a
INTRODUCTION The genes of the human a globin and globin loci are expressed in a specific temporal pattern, and in specific hemopoietic tissues during development. The embryonic globins (¢ and e) are expressed in the yolk sac blood islands until about the fifth week of gestation. At that time, adult globin (a) and fetal , globin (A,y and G^y) genes begin to be expressed in the liver. The liver is gradually replaced as the major site of hemopoiesis by the spleen and bone marrow, which express the adult globin genes (a, 6 and () (1). A similar developmental pattern of globin gene expression exists in mice. Hemopoiesis begins in the yolk sac at about day eight of gestation, and the fetal liver becomes the major site of hemopoiesis at about day ten. There is no fetal globin in mice, thus the mouse fetal liver produces only adult hemoglobins (2,3). Although the mechanisms that regulate developmental changes in globin gene expression are not completely understood, several lines of evidence indicate that two categories of DNase I hypersensitive sites (major and minor) are important in human a
*
To whom correspondence should be addressed
(x).
METHODS Transgenic Mice Transgenic mice were generated essentially as described by Hogan et al. (20). Embryos were obtained by breeding C57BL/6 x SJL F 1 hybrid parents. DNA was microinjected into
7094 Nucleic Acids Research, Vol. 18, No. 23 the pronuclei of one-cell embryos at a concentration of 2.0 nglpl. Injected embryos were cultured overnight in M 16 medium (20), and those that divided to two cells were transferred to the oviduct of a pseudopregnant female and allowed to develop to term. Transgenic animals were identified by Southern blot analysis of tail DNA, using a 4.8 kb genomic EcoRI fragment that spans the entire v gene as a probe. The probe fragment was labelled with 32p to a specific activity of 1I x 109 cpm/,ug using the random primer method (21).
Preparation of DNA for Microinjection An 882 bp fragment spanning HSII (17) was provided by Peter Curtin (University of California, San Francisco), and was inserted into the BamHI site of vector pUC 19. A 4.8 kb EcoRI fragment containing the v gene was isolated from cosmid pCL9, which contains the entire human a globin cluster (22), and was ligated into an EcoRI site located 21 bp from the HSII fragment. The 5' to 3' orientation of the r gene relative to the HSII fragment was the same as is found in the genome for the genes of the: globin cluster. For injection into mouse embryos, a 5.7 kb PvuI-HindIII fragment containing 777 bp of the original HSII insert linked to the r gene was isolated by preparative agarose gel electrophoresis. The fragment was recovered by electroelution, followed by concentration on an Elutip column (Schliecher & Schuell, Inc.) and ethanol precipitation. The DNA pellet was resuspended in injection buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) and filtered (0.2 Arm) prior to microinjection.
Preparation of Mouse RNA and DNA RNA was prepared as described by Chirgwin et al. (23). DNA was prepared by digestion of tissue with proteinase K, followed by chloroform extraction and ethanol precipitation. To obtain fetal and embryonic tissue samples, transgenic males were bred with normal females. The day that the copulation plug was observed was designated as day 0. Embryonic yolk sacs were collected at day 9 and fetal livers were collected at day 16. In each case, 10 or more tissue samples were pooled in the final RNA preparation. In some experiments RNA was prepared at day 16 from individual fetal livers, and DNA was prepared from the remainder of each fetus. Adult transgenic mice were treated with phenylhydrazine (25 ng/gm body weight injected intraperitoneally daily for 4 days) to induce production of nucleated red blood cells, and RNA was prepared from 200 1l of peripheral blood
units of M-MLV Reverse Transcriptase (Bethesda Research Laboratories). Reactions were incubated at 37°C for 1 hour. Nucleic acids were precipitated with ethanol, resuspended in denaturing sample buffer (95 % formamide) and analyzed on 8 % acrylamide/7M urea gels.
RESULTS In order to study the regulation of the human zeta gene during development, we established lines of transgenic mice and examined expression in staged embryos. The construct used to generate those mice is shown in figure 1. It consisted of a 4.8 kb DNA fragment containing the human v globin gene, linked to a 777 bp fragment spanning erythroid-specific hypersensitive site II (HSII). Three founder transgenic animals were identified by Southern blot hybridization, and were designated Z 1, Z5 and Zl1. To characterize the inheritance of the transgene, each of the founder animals was bred and offspring were screened by Southern blot hybridization. A typical result obtained using a human v probe is shown in figure 2. In DNA from the founder 0
10
30
20
v nom n I II I
50
40
60
70
80
90 VI
Cy Ay VP 1~~~~~~~~~~~~~~~~~~~~~1 I' 6
C
t030 kb
p
;
Human p Locus
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Human X Locus I,
HSII-Zeta Construct 0
1
2
3
4
5
6 kb
Figure 1. HSII-¢ construct. The human ce and /3 globin loci are shown, with the approximate positions of the genes represented by black boxes. The locations of the major DNaseI hypersensitive sites are indicated by arrows, and are identified by roman numerals. DNA fragments used to build the HSII-t construct are indicated by dotted lines, and features of the construct are represented as follows: HSII fragment, black line; ¢ exons, black boxes; ¢ introns, open boxes; ¢ 5' and 3' flanking sequences, hatched boxes.
-
on the fifth day.
Primer Extension Assay Oligonucleotide primers were 5'-end-labelled with 32p using T4 Kinase (Bethesda Research Laboratories), and were separated from unincorporated label using Nuctrap columns (Stratagene). The sequences of the primers were as follows: human ¢, 5'-TCTTGGTCAGAGACATGGCG-3'; mouse x, 5'-TGAGGGTTGGAGGCTGCGCT-3'; mouse x, 5'-CAGGCAGCCTTGATGTTGCTT-3'. Approximately 10,000 cpm of labelled primer was added to 10 Ag RNA. The RNA/primer mixture was evaporated to dryness and resuspended in 7.5 AI of 0.2M NaCl. Samples were sealed in glass microcapillaries, heated to 80°C for 2 minutes, and then incubated at 30°C for 1 hour. After hybridization, each sample was added to a tube containing a reverse transcription reaction mixture. The final reaction volume was 50 /d, and contained 50 mM Tris, pH 8.3, 30 mM NaCl, 75 mM KCl, 10 mM DTT, 3 mM MgCl2, 1 mM each dGTP, dATP, dCTP and TTP, 20 units of RNasin (Promega) and 200
.i.,
w
s
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Figure 2. Southern blot analysis of mice from the Z 11 transgenic line. DNA from the Z 11 founder (lane 1) and 5 offspring (lanes 2-6) was digested with EcoRI, blotted and probed with a 4.8 kb genomic DNA fragment spanning the human ¢ globin gene. Transgenic animals are characterized by a band at 4.8 kb that hybridizes to the human probe. Additional bands visible in lane I are the result of partial digestion of the DNA. Lanes 7 and 8 contain an amount of plasmid DNA equivalent to I and 2 copies of human ¢ per haploid genome respectively.
. wE
Nucleic Acids Research, Vol. 18, No. 23 7095
(ZI 1, lane 1), and in DNA from each of the transgenic offspring (lanes 2,4,5), there is a 4.8 kb fragment that hybridizes to the r probe. By comparing the intensity of the transgenic r signal to the intensity of the signal in lanes 7 and 8 which contain known amounts of r plasmid DNA, we estimate that the transgene copy number in the Z 11 line is -2-4 per haploid genome. Similarly, four of the eight offspring of Z5 were found to be transgenic, and the transgene copy number was estimated to be 1-2 per haploid genome. No transgenic offspring of Z1 were identified, and we presume that this animal is a mosaic. To verify that the entire HSII- construct was present in the Z5 and Z 11 lines, duplicate blots were probed for HSII. A band of the expected size was detected in each transgenic lane (data not shown). On the basis of these results, the Z5 and Z 11 transgenic lines were both chosen for further analysis. A primer extension assay was used to determine if the human v globin gene was expressed in the two transgenic lines, and at what stages of development. Total RNA was isolated from the yolk sacs of day 9 embryos and from the peripheral blood of adult transgenic animals. The embryonic tissues were derived from matings between transgenic males and normal females, and RNA was prepared from pools of 10 or more tissue samples to insure an adequate supply of material. Embryonic RNA samples were primed with a 1:1 mixture of the mouse x and human r primers. Adult RNA samples were primed independently with the mouse a primer and the mixed embryonic primers (x and O), because the ca and r extension products migrate at overlapping positions on the gel. The results of this experiment are shown in figure 3. In both transgenic lines, correctly initiated human r and mouse x transcripts are present in embryonic erythroid cells (lane 1). In adult erythroid cells, mouse a globin transcripts are present (lane 3), but human v and mouse x transcripts are undetectable (lane 2). The relative intensities of the primer extension bands approximate the relative amount of each transcript in the RNA sample. By assuming that 50% of the pooled tissue from which the embryonic RNA samples were prepared was transgenic, we estimate that human r transcripts are present at greater than 60% the level of mouse x transcripts in embryonic erythroid tissue of the Z5 line, and at greater than 30% the level of mouse x transcripts in embryonic tissue of the Z 11 line. The level of Z5
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human r and mouse x transcripts in transgenic adult RNA preparations is below the limit of detection of our assay. From this we conclude that transcription of the human r transgene is reduced to less than 1 % the level of the mouse a globin gene in adult animals. The mouse x globin gene is not expressed in the definitive erythroid cells of the fetal liver. To establish if there is a parallel reduction in the number of human r transcripts per animal at the same developmental stage, transgenic males were mated with normal females and total RNA from individual day 16 fetal livers was analyzed. DNA was isolated from each fetus, and transgenics were identified by Southern blot hybridization. Representative transgenic and non-transgenic RNA samples were assayed for the presence of transcripts from the mouse ae, the mouse x and the human r globin genes by primer extension. The results of this experiment are shown in figure 4. In both transgenic and control samples there is a strong mouse ax signal, and no detectable signal for mouse x or human t. Thus, transcription of the human r transgene has ceased in parallel with the switch from expression of murine embryonic to adult globin genes.
2 3
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Figure 3. Primer extension analysis of RNA from embryonic and adult erythroid tissues. In the panel on the left, RNA samples are from the Z5 line. In the panel on the right, RNA samples are from the Z 11 line. Lane 1 contains RNA prepared from embryonic yolk sacs, primed with a 1: 1 mixture of the human r and mouse x primers. Lane 2 contains adult RNA primed with a 1:1 mixture of the human ¢ and mouse x primers, and lane 3 contains adult RNA primed with the mouse ca primer.
primer
Mx
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Figure 4. Primer extension analysis of RNA prepared from individual fetal livers. In panel A, RNA samples are from the Z5 line. In panel B, RNA samples are from the Z1 1 line. The number above each lane identifies the RNA preparation used in that primer extension reaction. Transgenic samples are designated by a T and non-transgenic controls are designated by a C. The primer(s) used in each reaction are indicated below the lanes. Lane d9C contains RNA prepared from yolk sacs of transgenic embryos at day 9, primed with a 1:1 mixture of the mouse x and human r primers. Lane C contains the products of extension by all three primers with no added template RNA. Lane M contains a DNA size marker.
7096 Nucleic Acids Research, Vol. 18, No. 23
DISCUSSION We have constructed transgenic mice that harbor the human embryonic ¢ globin gene associated with a 777 bp DNA fragment that spans erythroid-specific DNase I hypersensitive site II from the distal 5' region of the globin gene cluster, and have examined the expression of the human gene during development. In six lines of transgenic mice that contained the same ¢ globin DNA fragment without HSII, no expression of the human gene was detected at any stage of development (E. R., unpublished). This result mirrors what is observed when human oa globin is introduced into mice without an enhancer (15,16). Using the HSII-D construct, we have observed correctly initiated transcription of the human gene in the erythroid cells of the embryonic yolk sac. We conclude that the embryonic gene, like the adult globin genes, requires the presence of an erythroidspecific enhancer for expression in transgenic mice. In addition, we find that the timing of expression of the human ¢ gene in erythroid tissues parallels that of the mouse x gene. From this we infer that like the murine embryonic globins, the human embryonic globin transgene is expressed only in the primitive erythroid cells of the yolk sac. The results of this study suggest that the 4.8 kb fragment containing the zeta globin gene with 0.55 kb of 5' and 2.75 kb of 3' flanking sequence contains all of the information necessary to specify that the gene be expressed in primitive erythroid cells and not be expressed in definitive erythroid cells. The evolutionary conservation of an approximately 300 bp region located immediately 5' to both the human r and the mouse x genes suggests that this noncoding sequence may have an important regulatory function (24). It is unlikely that expression of the human ¢ gene in murine erythroid cells is limited to a particular time in development as a direct result of its association with the HSII fragment. When the HSII fragment is linked to an adult human globin gene (a or () in transgenic mice, the adult gene can be expressed in adult tissues (17,18,19). Moreover, a human globin transgene in an HSLI- construct is not developmentally regulated; the adult gene is expressed in both primitive and definitive murine erythroid cells (E.S, K.A. and E.R., unpublished). Developmental regulation of the human r globin gene when linked to one of the major hypersensitive sites does not require the presence of other globin genes in cis. A similar observation was reported by Raich et al. (25), who found that developmental regulation of the human embryonic globin gene (e) is correct when that gene is linked to four major hypersensitive sites. In contrast, the loss of developmental regulation of human 'y and globin gene expression when the individual genes are introduced into mice linked to the major hypersensitive sites of the globin cluster is well documented (26,27,28). It has been suggested that the switch from fetal to adult globin gene expression involves a competition between the individual globin genes for interaction with the major hypersensitive sites (27,28). The outcome of this competition is thought to be influenced by positive and/or negative factors whose presence is developmentally regulated. According to this model, in the absence of competition the major hypersensitive sites form a stable interaction with a single gene and drive expression of that gene at all developmental stages (27,28). This model cannot account for the observed developmental regulation of embryonic globin transgenes, thus it seems likely that the mechanism for a
silencing expression of embryonic globin
genes at
later
stages
of development is fundamentally different from that which regulates the transition from fetal to adult gene expression.
Further gene transfer experiments will allow clarification of the role of proximal regulatory sequences and of their interactions with the major hypersensitive sites in the developmental regulation of globin gene expression. The mechanism of a globin gene switching during development has been refractory to experimental studies because of the lack of a cell culture or transgenic mouse system in which those genes were appropriately regulated. The use of constructs including major hypersensitive sites from the human j globin cluster has made it possible to obtain tissue-specific a globin expression (15,16), and our results suggest that this is a system in which the developmental regulation of expression of the a-like globin genes can also be studied. Because the murine pattern of embryonic a globin expression mirrors what occurs in humans, the transgenic mouse should provide an accurate and informative model system in which to study human a globin gene switching. After the submission of this manuscript Higgs et al. (29) reported the identification of sequences located far upstream of the human a globin genes that activate high level a globin expression both in MEL cells and in transgenic mice. The use of constructs containing those sequences, in place of the activating sequence from the 3 globin cluster, may provide additional insights into the requirements for a globin switching.
ACKNOWLEDGEMENTS We thank Shirley Clift and Deepa Kumar for identifying the zeta transgenic founders, and David Asarnow and Linda Couto for critical reading of the manuscript. This work was supported by NIH-HL20985 (EMR) and NRSA-HL07279 (EAS). This research was conducted at the Lawrence Berkeley Laboratory (Department of Energy Contract DE-AC03-76SF00098 to the University of California).
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