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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. In Vivo Search for Butyrate Responsive Sequences Using Transgenic Mice Carrying Gene Promoter Mutants By Betty S. Pace, Qiliang Li, and George Stamatoyannopoulos We describe an in vivo approach, in transgenic mice, aimed to identify promotar elements responsible for the induction of y globin expression by butyrate. Transgenic lines carrying human ‘ y gene promoter truncations at position -141, -201, -382, and -730 ‘y were treated with a amino butyric acid (aABA), and effects on y globin expression were anaRNA level. No induction of y gene l v e d at the m-nger expression was observed in animals carrying promoters truncated at positions -141, -201, or -382 ’y, suggesting either that butyrate responseelements (BREI are not located in the proximal y gene promoter or, if they were, they require the cooperation of upstream sequences for y gene induction. Two animals from one line carrying the -730 * y truncation responded to aABA treatment with significant increases in y gene expression, indicating that a BRE is located between position -382 and -730 region of the ‘ y gene promoter. Because the maximum induction by aABA is observed in transgenic mica carrying a ‘ y gene promoter extending to nucleotide -1350, it is likely that another butyrate responsive element is located between -730 and -1350 of the ‘y gene promoter. These results indicate that the transgenic mouse model can be used for identificationof DNA regions that contain ciselementsinvolved in y globin gen6 inducibility. 0 7996 b y The American Society of Hemstology. B miniosmotic pump with a capacity of 200 pL. The small capacity of the miniosmotic pump precluded the use of properly neutralized sodium butyrate, at the doses needed for our study. In contrast, aABA can be dissolved in normal saline, and the final volume of the solution containing the desired dose can be readily accommodated in the miniosmotic pump. aABA (Sigma Chemical CO,St Louis, MO), 250 mg/kg/d, was dissolved in normal saline and given subcutaneously over a 7-day period through a surgically implanted miniosmotic pump (Alza Corporation, Palo Alto, CA) according to the manufacturer’s specifications. Hematologic studies. Fifty microliters of blood was drawn from the retroorbital space of the transgenic mice for the analyses described below. The reticulum in the red blood cells was precipitated by incubating blood with 1% brilliant cresyl blue, and reticulocyte counts were calculated by counting 1,OOO cells. Measurements of F cells (hemoglobin F containing erythrocytes) were performed on methanol-fixed blood smears labeled by an indirect immunofluorescent assay using a monoclonal anti-y-chain antibody and anti-mouse F (ab‘)*-fluorescein isothiocyanate (Tag0 Diagnostic, Inc, Burlingame, CA). RNA analysis. Total cytoplasmic RNA was prepared by the method of Chomcynski and Sacchi.” RNA concentration and purity was assessed with UV spectrophotometry.” Globin messenger RNA (“A) was analyzed by RNase protection with the following probes: P T , ~(170). ~ linearized with BstEII, to give a 170-bp protected fragment, and pT,mousea (128) linearized with HindIII to give a 128-bp protected fragment. RNA (200 ng) was hybridized overnight at 45°C with lo6 cpm of each radiolabeled probe. After digestion with RNase A, the protected fragments were separated on a 6% polyacrylamide-8 m o m urea gel and autoradiographed without an intensifying screen. Human Ay and murine a “A was quanti- UTYRATE AND its analogues have been shown to induce embryonic globin gene expression in anemic chicken,’ to inhibit the y to p switch in sheep,’ and to induce fetal hemoglobin in baboon^,^" in transgenic mice carrying y globin gene constructs: and in a proportion of patients with sickle cell disease or homozygous Butyrate also influences the expression of several other genes.” It has been postulated that specific cis acting elements are responsible for the induction of gene expression by butyrate,” and a search for such elements has been done in various genes.”-’5Typically, the search for butyrate response elements (BRE) is done by transfecting recombinant genes into established cell lines and by measuring levels of gene expression after treatment with butyrate.”-LSIn this study, we search for BRE using an in vivo approach. Mice carrying various y gene promoter truncations were treated with a butyrate analogue, a amino butyric acid (aABA), which has been previously shown to induce y globin gene expression in human and baboon erythroid cells3as well as in transgenic mice carrying a Ay gene promoter extending to position -1350 If BRE were present along the y gene promoter, mice carrying truncated constructs lacking these elements were expected to fail to respond to aABA by increasing y gene expression. Mice carrying y gene promoters containing a BRE were expected to respond to butyrate treatment by increasing y gene expression. Our results suggest that this in vivo approach allows the identification of regions of the y gene promoter in which BRE are located. The approach may prove useful in the localization of cis sequences involved in induction of y globin genes by cytotoxic drugs or other compounds inducing fetal hemoglobin. MATERIALS AND METHODS * y gene constructs. A diagram illustrating the truncations of the Ay promoter used to make the recombinant DNA constructs is shown in Fig 1. All y gene fragments are linked to a p locus control region (pLCR) cassette containing core elements from DNAse I hypersensitive sites of the PLCR. All Ay gene fragments have the same 3’ end (the HindIII site) at position +1950. The 5’ ends are at positions -141, -201,-382, and -730 relative to the * y gene capsite. These constructs have been described previous1y.l’ Compounds. Induction of fetal hemoglobin in humans or baboons require the continuous intravenous administration of butyrate. Continuous infusion of drugs to the mouse is achieved by using a Blood, Vol88, No 3 (August I), 1996: pp 1079-1083 From the Department of Structural and Cellular Biology, University of South Alabama, Mobile; and the Department of Medicine, University of Washington, Seattle. Submitted June 14. 1995; accepted March 26, 19%. Supported by National Institutes of Health Grant HL 20899 and a Robert Woods Johnson grant to B.S.P. Address reprint requests to Betty S. Pace, MO, Department of Structural and Cellular Biology, MSB 2042, University of South Alabama, Mobile, AL 36688. The publication costs of this article were defiayed in part by page charge payment. This article must therefore be hereby marked “advdsement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. OOO6-4971/96/8803~0Q36$3.00/0 1079 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. ioao -1350 I PACE, LI, AND STAMATOYANNOPOULOS -7?0 -3p2 -20 1 Fig 1. pLCR”y promoter truncations used for production of transgenlc mice. Dlagramof the y globin promotershowing the position of transcriptionalmotifs and other regulatory elements. The 5’ end. of the “y promoter truncations ere indlcated with arrows and a number showing the site of truncation. -141 is an NCOI site, -201 is an Apa I site, -382 is a shc I site, -730 is a Ssp I site, and -1350 is a H/ndlll site. Each of the five constructstested contain the 2.5 kb pLCR ceuette and the human ”y globin gene (with a 3’ end at position +1950 ~Hhdlll&e)relative to the cap site). 8 corresponds to a region containing e putetlve negativeelement identifiedwith studies in transgenic mice.” tated using a Molecular Dynamics PhosphorImager (Sunnyvale, CA). Human y globin gene expression per copy of the transgene and per copy of the endogenous murine a gene was calculated as %ylMocu/copy. RESULTS Experimental approach. The truncations of the y globin promoter used to make the five constructs are shown in Fig 1. Animals were treated with a continuous infusion of aABA, 250 mgkg/d, for 7 days using a miniosmotic pump. Peripheral blood samples were analyzed for y mRNA production, and F cells were measured using a fluorescent antiy chain antibody. y mRNA levels were expressed as a percentage of murine a mRNA, after correction for the expression per copy of the transgene and per copy of the endogenous murine a gene. A consistent increase in y mRNA production during several days of treatment was scored as a positive response to butyrate treatment. Lack of an increase in y mRNA production was not automatically scored as a negative response because it could be due to a lack of biological effect of butyrate in the treated animals. As an independent measure of biological effect of butyrate in such animals, we used the property of aABA to stimulate erythropoiesis and induce reticulocytosis. Reticulocytes were counted for several days after treatment, and a positive response to aABA was scored when the level of peak reticulocytosis was 50% higher than the baseline pretreatment value. Mice carrying -141 *y constructs. Truncation of the Ay promoter at position -141 conserves all of the essential elements of the promoter (the duplicated CAAT box, TATA box, etc) but it destroys the CACC site around nucleotide -141. These mice express y globin in the fetal stage of development but lack y expression in the adult stage, suggesting that a negative element, perhaps the site of binding for y gene repressors, is located 3‘ to position -141 in the Ay gene promoter.” cis elements in this region of the y promoter previously shown to be involved in y gene developmental regulation include the distal CAAT box that is mutated in the - 117AyGreek type of Hereditary Persistence of Fetal Hemoglobinzo.zland a “stage selector element” located in the -50 Ay region.” Because the Ay gene of the -141 Ay construct is “on” in the transgenic fetuses, it was reasonable to ask whether treatment of adult mice with butyrate will result in induction of y gene expression. Six animals from three lines carrying this construct were used. Treatment with aABA increased reticulocytes about fourfold in animal 2 of line 134/1-5, 1.8-fold in animal 1 of line 134/1-5, and 2.6-fold in animal 1 of line 134/5-6. In these animals, pretreatment y mRNA levels were lower than 1% of murine a mRNA and essentially remained unchanged (Table 1). These results suggest either that no cis elements that can respond to in vivo administration of butyrate are located 3‘ to nucleotide - 141 of the Ay promoter or that the lack of response is due to the fact that the “ y gene of the - 141 Ay mice is silenced in the adult. Mice carrying -201 Ay or -382 Ay constructs. Addition to the - 141 “ y construct of the sequence between -141 and -201 of the Aypromoter results in the loss of developmental regulation so that the level of y gene expression in adult animals is equal to that of the fetuses. Therefore, transgenic mice carrying a y gene promoter extending to position -201 Ayor -382 Ay display abundant y gene expression. Furthermore, in these animals, there is an excellent correlation between level of y gene expression and the number of the integrated transgenes indicating that y gene expression is Table 1. y Globin Gane Induction After Treatment With aABA Transgenic M i Carrying the pLCR -141”y Construct YlMOUl Transgenic Animal Animal 13411-5 1 Copy Day of No. Treatment 2 0 6 7 8 9 13411-5 2 2 0 6 7 a 13411-5 3 2 9 0 6 7 a 13413-2 13415-6 1 1 49 8 9 0 5 7 8 9 0 6 7 a 134/5-6 2 8 9 0 6 7 a 9 Reticulocytes (%) F Cells 1%) 2.5 3.5 4.2 4.5 4.2 1.8 4.8 7.5 5.7 6.1 2.3 1.5 3.9 1.5 5.6 2.6 2.0 4.0 4.4 4.2 2.2 3.7 4.4 5.3 5.8 3.0 5.1 3.0 5.4 5.6 0.6 1.7 1.5 1 .o 1 .o 0.3 0.2 0.8 0.5 0.2 0.3 0.6 0.2 0.4 0.8 Not done 1.7 0.6 0.7 0.8 0.9 0.1 0.2 0.1 0.1 0.1 COPY (%) 0.08 0.07 0.08 0.14 0.12 0.20 0.20 0.20 0.17 0.24 0.10 0.14 0.18 0.12 0.10 0.49 0.50 0.53 0.43 0.19 0.31 - 0.20 0.20 0.43 0.52 0.56 0.52 0.50 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1081 IN VIVO SEARCH FOR BRE copy-number dependent.17 These results have been interpreted to suggest that the -141 to -201 region of the Ay promoter represents the site of interaction between the promoter and the LCR.I7If a cis element responsive to butyrate is placed 3’ to -201 Ay, one would expect that y gene expression will be induced when the -201 Ay or the -382 Ay transgenic mice are treated with a-ABA. Two lines carrying the -201 Ay construct were used. The baseline y mRNA level of line 136/3-4 was 25.5%of murine a. Treatment with aABA increased reticulocytes by about 1 J-fold, but there was no further increase in y mRNA. The mouse from line 136/4-2 had a baseline y mRNA level 17.4%of murine a;there was no y gene induction although aABA stimulated production of reticulocytes (from a baseline value of 5% to a peak value of 8.5%) (Table 2). The three lines carrying the -382 Ay promoter construct (Table 3) responded to aAJ3A with consistent increases in reticulocyte production (from 6.1% to 9.0%, line 135/1-2; from 3.0%to 7.3% line 1394-2; from 4.0% to 6.0% line 135/4-8). Two lines (135/1-2 and 135/4-2) failed to change y gene expression during treatment (Table 3). y mRNA increased in line 135/4-8 from 18.9%to 29.2% on treatment day 5, but it was 22.8% on day 6 and 23.1% on day 7, suggesting that the higher y mRNA level of day 5 was due to incorrect measurement. Transgenic mice carrying the -730 A y construct. Extension of the Ay gene promoter to the -730 Ay site results in two changes in Ay gene expression. First, Ay gene expression becomes copy number independent, so that the excellent correlation between the copy number of the transgene and the level of Ay gene expression that is characteristic of the -201 Ay and the -382 Ay transgenic mice disappear^.'^ This phenomenon indicates that the -382 to -730 sequence renders the ’7 promoter sensitive to position effects. Second, in several -730 Ay transgenic lines, there is developmental regulation of y gene expression (ie, high Ay gene expression in the fetus but very low expression in the adult).I7 These observations were interpreted to indicate that the -382 to -730 region contains a sequence that acts as a silencer, hence the low y globin expression in the adult animals of certain lines. Eight transgenic animals from four lines were used (Table Table 2. y Globin Gene Induction After Treatment With aABA Transgenic Mice Carrying the pLCR -2OlAy Construct F COPY Transgenic Line Animal No. pLCR-20lAy 13613-4 1 5 Day of Treatment Reticulocytes (%) 0 6 7 2.6 1.6 3.4 4.0 4.4 5.0 4.2 8.5 4.4 5.9 8 13614-2 1 3 9 0 6 7 8 9 Cells ylMout Copy (461 (%) 90 89.5 25.5 15.0 20.4 23.9 22.3 17.4 15.9 20.3 18.4 16.4 Table 3. y Globin Gene Induction Aftor Treatment With aABA Transgenic Mice Carrying the pLCR - e y Construct YmW Transgenic Line pLCR-382Ay 13511-2 Animal Copy No. Day of Treatment 1 6 0 6 7 Reticulocytes F Calls (%J (%) 8 13514-2 1 5 13514-8 1 3 9 0 5 6 7 8 0 5 6 7 6.1 8.9 9.0 6.5 6.0 3.0 7.3 5.0 2.6 3.7 4.0 4.7 5.5 6.0 90.4 88.5 90 Copy 1%) 9.3 - 7 8.1 10.2 15.9 16.1 16.1 17.2 17.4 18.9 29.2 22.8 23.1 4). Line 137/1-2 has barely detectable y gene expression in the adult; the two animals of this line failed to respond to butyrate treatment by increasing y mRNA (although reticulocytosis occurred in one animal). y mRNA levels in line 137/2-4 range from 1.5%to 1.8% of murine a. Animal 1 of this line responded to butyrate with brisk reticulocytosis but increased y mRNA only slightly over baseline levels. aABA did not induce significant reticulocytosis in animal 2. y mRNA levels in line 137/5-2 is 6.4% of murine a;this line failed to respond to butyrate treatment (Table 4). The three animals from line 137/5-1 had y mRNA levels of 5.5%, 12.8%, and 16.7% of murine a.The study of animal 1 was incomplete because “A measurements were done only on days 6 and 7; y mRNA did not increase above the baseline level in these 2 days. Animal 2 increased y mRNA from a baseline value of 12.8%to 22.4% by day 9 (Table 4 and Fig 2). Animal 3 increased y mFWA from baseline value of 16.7%to 51.1% by day 9 (Fig 2). DISCUSSION It has been previously shown that transgenic mice carrying a y gene construct with Ay promoter extending to position -1350 Ay increase y gene expression in response to treatment by 5 azacytidine, butyrate, erythropoietin, hydroxyurea, or Didox, an inhibitor of ribonucleotide red~ctase.’*~.*~ The molecular mechanisms whereby these compounds induce y globin expression remain unknown. 5-Azacytidine presumably demethylates regulatory sequences, thus modulating the interaction between these sequences and DNA binding proteins. A likely mechanism of action of the other hemoglobin (Hb)F-inducing agents is through modulation of proteins that interact with DNA regulatory sequences. Nothing is known about the regulatory sequences of the y globin gene through which these Hb F inducers presumably exert their effects. In this report, we test whether regions containing such sequences can be recognized with in vivo studies in transgenic mice. Truncations of the Ay gene promoter at position -141, From www.bloodjournal.org by guest on August 3, 2017. For personal use only. PACE, LI, AND STAMATOYANNOPOULOS 1082 Table 4. y Globin Gene Induction After Treatment With aABA Transgenic Mice Carrying the pLCR -73Py Construct Transgenic Line 13711-2 13711-2 13712-4 13712-4 13715-2 13715-1 13715-1 13715-1 Animal Copy No. F y/Moa/ Day of Reticulocytes Cells Treatment (%) (%) Copy I%) 0 4 5 6 1 0 6 3.9 3.5 3.6 3.4 4.5 2.8 2.6 2.6 4.2 4.2 3.1 8.7 1.3 7.2 4.5 3.7 Rare Rare 0.1 Rare Rare 0.3 0.8 0.1 0.8 2.0 Rare Rare Rare Rare Rare 0.5 5.9 0.1 1 8 9 0 4 5 6 7 0 6 7 8 9 0 6 1 8 9 0 6 7 0 6 1 8 9 0 6 1 8 9 4.3 2.1 6.3 7.0 6.4 2.4 3.5 3.8 2.0 3.1 4.4 5.3 5.8 1.9 2.3 2.5 4.4 10.9 90 90 90 90 0.04 0.02 0.05 0.04 0.03 0.10 0.09 0.12 0.04 0.04 1.8 2.4 2.5 1.7 2.3 1.5 1.o 1.5 1.o 1.o 6.4 5.0 6.7 6.7 7.0 5.5 4.0 3.0 12.8 14.1 14.1 18.4 22.4 16.7 23.5 30.6 31.5 51.1 they require the cooperation of upstream elements to elicit a response to butyrate. Previous evidence suggests that the -382 to -730 region of the Ay promoter contains a y gene silencer that is sensitive to “position effects” (ie, its action depends on the position of integration of the transgene). The mechanism of the position-dependence of this element remains speculative. Perhaps the -382 to -730 Ay silencer acts by influencing the conformation of the chromatin of the upstream y promoter in a manner inhibiting the interaction between y promoter and the LCR. Under this hypothesis, “position effects” arise because the position of integration of the transgene affects the conformation of this region and secondarily influences the interaction between the silencer and the LCR. Of the four -730 Ay transgenic lines treated with aABA, only two animals of one line responded by increasing y gene expression by twofold to threefold. The fact that even two animals responded to butyrate with significant increases in y mRNA levels suggests that a BRE is located in the -382 to -730 Ay region. The lack of response in the other lines can be attributed to position effects. Butyrate may activate y globin expression by modifying DNA binding proteins and the interaction of such proteins with the y silencer may be modu- A 30 B 601 -201 or -382 abolish the ability of butyrate to induce y gene expression. These results make it unlikely that a butyrate responsive element is located 3’ to nucleotide -382 of the Ay gene promoter. It could be argued, however, that the lack of y gene induction in these mice is not due to absence of a BRE in the proximal promoter but to the fact that the Ay genes of the -201 and -382 animals have reached a maximal level of expression and aABA could not induce y gene expression further. This is not a strong argument, however, because levels of Ay gene expression higher than those of -201 Ay, and -382 * y mice have been reached when animals carrying a promoter extending to -730 Ay (animal 137/51-3 of Table 4) or to -1350 Ay’6 were treated with butyrate. The lack of induction of the y gene of the -201 Ay and -382 transgenic mice suggests either that the y globin BRE are located upstream of position -382 of the Ay promoter or that if BRE are located 3‘ to position -382, 1371 5-11 2 1 13715-113 Days of Treatment Fig 2. Induction of ‘ y mRNA production in two pLCR -730 ‘y mice treated with oABA. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. IN VIVO SEARCH FOR BRE lated depending on the position of integration of the transgene. The highest degree of induction of gene expression by butyrate has so far been observed in transgenic mice that carry a Ay gene construct extending to position -1350 in the promoter?*16Although only one such pLCR line with promoter extending to - 1350 is available, multiple generations of this line have been treated with butyrate and induction of y globin expression has been consistently ob~ e r v e d . ~These . ' ~ results suggest that another BRE is located between positions -730 and - 1350of the * y gene promoter. Our results demonstrate that in vivo studies in transgenic mice allow identification of promoter regions that are involved in butyrate inducibility. This approach can be used in the delineation of the cis elements involved in the induction of hemoglobin by other agents, such as hydroxyurea, erythropoietin, or 5-azacytidine. Although the in vivo approach we describe here allows the localization of regions containing BRE, further use of transgenic mice for pinpointing the structure of the localized BRE is prohibited by the expense of producing and maintaining transgenic mice. Studies using stable or transient expression systems are more appropriate for that purpose. Once a BRE sequence has been identified with in vitro assays, studies in transgenic mice can be used to verify the in vivo relevance of the in vitro findings. ACKNOWLEDGMENT We thank Bonnie Lenk and Shem Brenner for their help in the preparation of this manuscript. REFERENCES 1. Ginder GD, Whitters MJ, Pohlman J K Activation of a chicken embryonic gene in adult erythroid cells by 5-azacytidine and sodium butyrate. Roc Natl Acad Sci USA 81:3954, 1984 2. Perrine SP, Rudolph A, Faller D V Butyrate infusions in the ovine fetus delay the biologic clock for globin gene switching. hoc Natl Acad Sci USA 85:8540, 1988 3. Constantoulakis P, Papayannopoulou T, Stamatoyannopoulos G: a-Amino-N-butyric acid stimulates fetal hemoglobin in the adult. Blood 72:1961, 1988 4. Constantoulakis P, Knitter G, Stamatoyannopoulos G: On the induction of fetal hemoglobin by butyrates: In vivo and in vitro studies with sodium butyrate and comparison of combination treatment with 5-AzaC and AraC. Blood 74:1963, 1989 5. Constantoulakis P, Josephson B, Mangahas L, PapayannopouIOU T: Locus control region-A y transgenic mice: A new model for studying the induction of fetal hemoglobin in the adult. Blood 77:1326, 1991 6. Pemne SP, Ginder GD, Faller DV, Dover GJ, Ikuta T, Witkowska HE, Cai S, Vichinsky EP, Olivieri N F A short-term trial of butyrate to stimulate fetal-globin-gene expression in the 0-globin disorders. N Engl J Med 328:81, 1993 7. Dover GJ, Brusilow S, Samid D: Increased fetal hemoglobin 1083 in patients receiving sodium 4-phenylbutryate. N Engl J Med 327569, 1992 8. Sher GD, Ginder GD, Little J, Yang S, Dover GJ, Olivier NF: Extended therapy with intravenous arginine butyrate in patients with p hemoglobinopathies. N Engl J Med 332:1606, 1995 9. Perrine S, Pekatos P, Faller DV, Bergsagel J, Ikuta T, Dover G, Phipps K, Foumarakis B, Boosalis V, Stamatoyannopoulous G, Brauer M, Huerta P, Cao M, Travis M, Atweh G F Hematological efficacy and elimination of transfusion requirements in thalassemia major by intermittent low-dose butyrate therapy. Blood 86:482, 1995 (abstr) 10. Kruh J: Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem 42:65, 1982 11. Glauber JG, Wandersee NJ, Little JA, Ginder GD: 5'-Flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells. Mol Cell Biol 11:4690, 1991 12. Bohan C, York D, Srinivasan A: Sodium butyrate activates human immunodeficiency virus long terminal repeat -directed expression. Biochem Biophys Res Commun 148:899, 1987 13. Fregeau CJ, Helgason CD, Bleackley RC: Two cytotoxic cell proteinase genes are differentially sensitive to sodium butyrate. Nucleic Acids Res 20:3113, 1992 14. Gill RK, Chistakos S: Identification of sequence elements in mouse calbindin-Dz8kgene that confer 1,25-dihydroxyvitamin D,and butyrate-inducible responses. Proc Natl Acad Sci USA 902984, 1993 15. Knochbin S, Wolffe A P Developmental regulation and butyrate-inducible transcription of the Xenopus histone H1 promoter. Gene 128:173, 1993 16. Pace B, Li Q, Peterson K, Stamatoyannopoulos G: a-Amino butyric acid fails to reactivate the totally silenced y gene of the 0 locus YAC. Blood 84:4344, 1994 17. Stamatoyannopoulos G, Josephson B, Zhang J, Li Q: Developmental regulation of human y globin genes in transgenic mice. Mol Cell Biol 13:7636, 1993 18. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156, 1987 19. Current Protocols in Molecular Biology. New York, NY, Wiley, 1991 20. Gelinas R, Endlich B, Pfeiffer C, Yegi M, Stamatoyannopoulos G: G to A substitution in the distal CCAAT box of the Ayglobin gene in Greek hereditary persistence of fetal haemoglobin. Nature 313:323, 1985 21. Collins FS, Metherall JE, Yamakawa M, Pan J, Weissman SM, Forget BG: A point mutation in the Ag-globin gene promoter in Greek hereditary persistence of fetal haemoglobin. Nature 313:325, 1985 22. Jane SM, Ney PA, Vanin EF, Gumucio DL, Nienhuis AW: Identification of a stage selector element in the human y-globin gene promoter that fosters preferential interaction with the 5' HS2 enhancer when in competition with the 0-promoter. EMBO J 11:2961, 1992 23. Pace B, Elford H, Stamatoyannopoulos G: Transgenic mouse model of pharmacologic induction of fetal hemoglobin: Studies using a new ribonucleotide reductase inhibitor, Didox. Am J Hematol 45:136, 1994 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 1996 88: 1079-1083 In vivo search for butyrate responsive sequences using transgenic mice carrying A gamma gene promoter mutants BS Pace, Q Li and G Stamatoyannopoulos Updated information and services can be found at: http://www.bloodjournal.org/content/88/3/1079.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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