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Effect of trans8, cis10+cis9, trans11 Conjugated Linoleic Acid Mixture on Lipid Metabolism in 3T3-L1 CellsShama V. JosephDepartment of Food Science and Nutrition, Institute of Nutraceuticals and Functional FoodsLaval UniversityJessica R. MillerDepartment of Biochemistry and Molecular BiologyDalhousie UniversityRoger S. McLeodDepartment of Biochemistry and Molecular BiologyDalhousie UniversityHélène JacquesDepartment of Food Science and Nutrition, Institute of Nutraceuticals and Functional FoodsLaval UniversityOriginal ArticleDOI:
10.-009-3309-3Cite this article as: Joseph, S.V., Miller, J.R., McLeod, R.S. et al. Lipids (3. doi:10.-009-3309-3Evidence suggests that minor isomers of conjugated linoleic acid (CLA), such as trans8, cis10 CLA, can elicit unique biological effects of their own. In order to determine the effect of a mixture of t8, c10+c9, t11 CLA isomers on selected aspects of lipid metabolism, 3T3-L1 preadipocytes were differentiated for 8 days in the presence of 100 μM linoleic acid (LA); t8, c10+c9, t11 CLA; t10, c12+c9, t11 CLA or purified c9, t11 CLA. Whereas supplementation with c9, t11 and t10, c12+c9, t11 CLA resulted in cellular triglyceride (TG) concentrations of 3.4 ± 0.26 and 1.3 ± 0.11 μg TG/μg protein, respectively (P & 0.05), TG accumulation following treatment with CLA mixture t8, c10+c9, t11 was significantly intermediate (2.5 ± 0.22 μg TG/μg protein, P & 0.05) between the two other CLA treatments. However, these effects were not attributable to an alteration of the Δ9 desaturation index. Adiponectin content of adipocytes treated with t8, c10+c9, t11 mixture was similar to the individual isomer c9, t11 CLA, and both the t8, c10+c9, t11 and c9, t11 CLA groups were greater (P & 0.05) than in the t10, c12+c9, t11 CLA group. Overall, these results suggest that t8, c10+c9, t11 CLA mixture affects TG accumulation in 3T3-L1 cells differently from the c9, t11 and t10, c12 isomers. Furthermore, the reductions in TG accumulation occur without adversely affecting the adiponectin content of these cells.Conjugated linoleic acidIsomersTriglyceridesAdiponectin3T3-L1 cellsBSABovine serum albuminCLAConjugated linoleic acidDMEMDulbecco’s Modified Eagle’s MediumFAMEFatty acid methyl esterFFAFree fatty acidGC-FIDGas chromatograph(y)-flame ionisation detectorLALinoleic acidMUFAMonounsaturated fatty acidNLNeutral lipidSCDStearoyl CoA desaturaseTGTriglycerideTLCThin layer chromatography1.Belury MA (2002) Dietary conjugated linoleic acid in health: physiological effects and mechanisms of action. Annu Rev Nutr 22:505–5312.Park Y, Albright KJ, Liu W, Storkson JM, Cook ME, Pariza MW (1997) Effect of conjugated linoleic acid on body composition in mice. Lipids 32:853–8583.Ostrowska E, Muralitharan M, Cross RF, Bauman DE, Dunshea FR (1999) Dietary conjugated linoleic acids increase lean tissue and decrease fat deposition in growing pigs. J Nutr 29:4.Wahle KW, Heys SD, Rotondo D (2004) Conjugated linoleic acids: are they beneficial or detrimental to health? Prog Lipid Res 43:553–5875.Pariza MW (2004) Perspective on the safety and effectiveness of conjugated linoleic acid. Am J Clin Nutr 79:1132S–1136S6.Flowers MT, Miyazaki M, Liu X, Ntambi M (2006) Probing the role of steroyl-CoA desaturase-1 in hepatic insulin resistance. J Clin Invest 116:7.Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski CM, Yandell BS, Song Y, Cohen P, Friedman JM, Attie AD (2002) Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity. Proc Natl Acad Sci USA 99:18.Gutierrez-Juarez R, Pocai A, Mulas C, Ono H, Bhanot S, Monia BP, Rossetti L (2006) Critical role of stearoyl-CoA desaturase-1 (SCD1) in the onset of diet-induced hepatic insulin resistance. J Clin Invest 116:9.Park Y, Storkson JM, Ntambi JM, Cook ME, Sih CJ, Pariza MW (2000) Inhibition of hepatic stearoyl-CoA desaturase activity by trans-10, cis-12 conjugated linoleic acid and its derivatives. Biochim Biophys Acta 210.Wendel AA, Belury MA (2006) Effects of conjugated linoleic acid and troglitazone on lipid accumulation and composition in lean and Zucker diabetic fatty (fa/fa) rats. Lipids 41:241–24711.Choi Y, Kim YC, Han YB, Park Y, Pariza MW, Ntambi JM (2000) The trans-10, cis-12 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes. J Nutr 130:12.Choi Y, Park Y, Pariza MW, Ntambi JM (2001) Regulation of stearoyl-CoA desaturase activity by the trans-10, cis-12 isomer of conjugated linoleic acid in HepG2 cells. Biochem Biophys Res Commun 284:689–69313.Havel PJ (2004) Update on adipocyte hormones: regulation of energy balance and carbohydrate/lipid metabolism. Diabetes 53:S143–S15114.Perfield JW, Saebo A, Bauman DE (2004) Use of conjugated linoleic acid (CLA) enrichments to examine the effects of trans-8, cis-10 CLA, and cis-11, trans-13 CLA on milk-fat synthesis. J Dairy Sci 87:15.Reany MJT, Liu Y, Westcott ND (1999) Advances in conjugated linoleic acid research, vol 1. AOCS Press, Champaign16.Destaillats F, Japiot C, Chouinard PY, Arul J, Angers P (2005) Short communication: rearrangement of rumenic acid in ruminant fats: a marker of thermal treatment. J Dairy Sci 88:17.Ostrowska E, Cross RF, Muralitharan M, Bauman DE, Dunshea FR (2003) Dietary conjugated linoleic acid differentially alters fatty acid composition and increases conjugated linoleic acid content in porcine adipose tissue. Br J Nutr 90:915–92818.Bissonauth V, Chouinard PY, Marin J, Leblanc N, Richard D, Jacques H (2008) Altered lipid response in hamsters fed cis-9, trans-11+trans-8, cis-10 conjugated linoleic acid mixture. Lipids 43:251–25819.Bissonauth V, Chouinard Y, Marin J, Leblanc N, Richard D, Jacques H (2006) The effects of t10, c12 CLA isomer compared with c9, t11 CLA isomer on lipid metabolism and body composition in hamsters. J Nutr Biochem 17:597–60320.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–27521.Hara A, Radin NS (1978) Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem 90:420–42622.Hashimoto N, Aoyama T, Shiori T (1981) New methods and reagents in organic synthesis: a simple efficient preparation of methyl esters with trimethylsilyldiazomethane (TMSCHN2) and its application to gas chromatographic analysis of fatty acids. Chem Pharm Bull 29:23.Yurawecz MP, Kramer JK, Ku Y (1999) Methylation procedures for conjugated linoleic acid. AOCS Press, Champaign24.MacDougald O, Lane M (1995) Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345–37325.Evans M, Lin X, Odle J, McIntosh M (2002) Trans-10, cis-12 conjugated linoleic acid increases fatty acid oxidation in 3T3-L1 preadipocytes. J Nutr 132:450–45526.Miller JR, Siripurkpong P, Hawes J, Majdalawieh A, Ro HS, McLeod RS (2008) The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARgamma-dependent and PPARgamma-independent mechanisms. J Lipid Res 49:550–56227.Brown M, Evans M, McIntosh M (2001) Linoleic acid partially restores the triglyceride content of conjugated linoleic acid-treated cultures of 3T3-L1 preadipocytes. J Nutr Biochem 12:381–38728.Evans M, Park Y, Pariza M, Curtis L, Kuebler B, McIntosh M (2001) Trans-10, cis-12 conjugated linoleic acid reduces triglyceride content while differentially affecting peroxisome proliferator activated receptor gamma2 and aP2 expression in 3T3-L1 preadipocytes. Lipids 36:29.Brown JM, Halvorsen YD, Lea-Currie YR, Geigerman C, McIntosh M (2001) Trans-10, cis-12, but not cis-9, trans-11, conjugated linoleic acid attenuates lipogenesis in primary cultures of stromal vascular cells from human adipose tissue. J Nutr 131:30.Granlund L, Larsen LN, Nebb HI, Pedersen JI (2005) Effects of structural changes of fatty acids on lipid accumulation in adipocytes and primary hepatocytes. Biochim Biophys Acta 31.Brown JM, McIntosh MK (2003) Conjugated linoleic acid in humans: regulation of adiposity and insulin sensitivity. J Nutr 133:32.Brown JM, Boysen MS, Jensen SS, Morrison RF, Storkson J, Lea-Currie R, Pariza M, Mandrup S, McIntosh MK (2003) Isomer-specific regulation of metabolism and PPARgamma signaling by CLA in human preadipocytes. J Lipid Res 44:33.Ntambi JM, Miyazaki M (2003) Recent insights into stearoyl-CoA desaturase-1. Curr Opin Lipidol 14:255–26134.Pischon T, Rimm EB (2006) Adiponectin: a promising marker for cardiovascular disease. Clin Chem 52:797–79935.Fu Y, Luo N, Klein RL, Garvey WT (2005) Adiponectin promotes adipocyte differentiation, insulin sensitivity, and lipid accumulation. J Lipid Res 46:36.Poirier H, Shapiro JS, Kim RJ, Lazar MA (2006) Nutritional supplementation with trans-10, cis-12-conjugated linoleic acid induces inflammation of white adipose tissue. Diabetes 55:37.Cooper M, Miller JR, Mitchell PL, Currie DL, McLeod RS (2008) Conjugated linoleic acid isomers have no effect on atherosclerosis and adverse effects on lipoprotein and liver lipid metabolism in apoE(-/-) mice fed a high-cholesterol diet. Atherosclerosis 200:294–302
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Different integration site structures between L1 p
官方公共微信&&&trans cis
的翻译结果:
查询用时：0.158秒
&在分类学科中查询
Synthesis of
cis,trans,cis 1,2,3,4Cyclobutanetetracarboxylic Dianhydride
顺,反,顺-1,2,3,4-环丁烷四甲酸二酐的合成研究
The crystal structure of 2a was determined. The double function of 2a,
isomerization of the azo group, and the torsion angle between the benzene and the coumarin ring suggested that azocoumarins might be uesful as efficient molecular devices.
同时用X射线单晶衍射测定了化合物 2a的晶体结构 ,其特殊的双功能分子键连、偶氮基的顺反异构和苯环与香豆素环平面间的扭转夹角 ,使之可能成为有效的分子器件材料
E-4,5-Disubstituted-2-Pyrrolichinones was synthesized under phase transfer catalysis,trans:cis was 97:3,The yield of the product was 74%,Structures of product was confirmed by
1 HNMR and MS.
采用PTC法合成了E - 4,5 -二取代苯基 - 2 -吡咯烷酮 ,反式异构体与顺式异构体之比为 97∶3,产率高达 74% ,其结构用1HNMR和MS表征。
Disubstituted
Pyrrolichinones was highly stereoselective synthesized under phase transfer catalysis, trans:cis was 97:3; the yield of the product was 74%; structures of product was confirmed by HNMR and MS.
采用PTC法高立体选择性合成了E 4.5
吡咯烷酮 ,反式异构体与顺式异构体之比为 97∶3,产率高达 74% ,其结构用1HNMR和MS表征 .
Lipoxygenase (LOX) contains a nonheme iron in it’s catalytic center. It can catalyze the oxidation of polyunsaturated fatty acids containing the cis,cis-1,4-pentadiene moiety to the corresponding hydroperoxides (HPOD) of conjugated (trans, cis)-dienes, which are important intermediates of synthesizing drugs and chemicals.
脂肪氧合酶(LOX)的结构中含有非血红素铁,能专一催化具有cis,cis-1,4-戊二烯结构的多元不饱和脂肪酸加氧形成具有共轭双键的脂肪酸氢过氧化物(HPOD)。
Lipoxygenase (LOX) contains a nonheme iron in its catalytic center. It can catalyze the oxidation of polyunsaturated fatty acids containing cis,cis-1,4-pentadiene moiety to the corresponding hydroperoxides (HPOD) of conjugated (trans, cis)-dienes, which are important intermediates for synthesizing drugs and chemicals.
脂氧酶(LOX)的结构中含有非血红素铁,能专一催化具有cis,cis-1,4-戊二烯结构的多元不饱和脂肪酸加氧形成具有共轭双键的脂肪酸氢过氧化物(HPOD)。
From 1982 to 1984 for monitoring a sexual lure reagent for this specie (trans, cis) -3, 13 octadecadienol was used during the emerging stage. It is possible to predict, in Beijing district, that the initial stage of emergence is in first ten days of May, to the middle of December. This stage is prolonged for 5 months.
年采用白杨透翅蛾性引诱剂反3、顺13-十八碳二烯醇对成虫发生期进行监测,可准确地预测成虫发生期在北京地区5月上旬为成虫羽化初期,直至10月中旬结束,历期可长达5个月之久,并测到有第二代成虫的出现。
CIS-TRANS PHOTOISOMERIZATION OF OCIMENE
罗勒烯的光化学顺反异构化反应
The cis-trans configuration of the compounds was analysed.
对化合物的顺反构型作了分析。
查询“trans cis”译词为用户自定义的双语例句&&&&我想查看译文中含有：的双语例句
为了更好的帮助您理解掌握查询词或其译词在地道英语中的实际用法，我们为您准备了出自英文原文的大量英语例句，供您参考。&&&&&&&&&&&& Azoxybisbenzo-15-crown-5 was synthesized.Photoinduced trans-cis iso-merization as well as its ability to extract alkali metals and rare earth metals' were dis-cussed. 合成了一种新的双冠醚,氧化偶氮双苯并15-冠-5。初步探讨了它的光异构现象及其对稀土和碱金属离子的萃取能力。 The catalysis of trans-cis isomerizationof trans-diaquobis (oxalato) -chromate(Ⅲ) byCoSO_4 was investigated at ionic strength 1.5mol·L~(-1), pH 3 and 25, 35 and 45℃, respe-ctively.Linear behavior is observed betweenthe rate and the concentration of Co~(2+) ionuntil ca. 0. 15 mol·L~(-1), after the ratebecomes much faster. If no CoSO_4 ion pair catalysis exists,the rate constant of isomerization k_(obs) willhave the formk_(obs)=k_(H_2O)+k_(Na~+)[Na~+] + k_(H~+) [H~+] ++ k_(Co~(2+)[Co~(2+)]= k_1+... The catalysis of trans-cis isomerizationof trans-diaquobis (oxalato) -chromate(Ⅲ) byCoSO_4 was investigated at ionic strength 1.5mol·L~(-1), pH 3 and 25, 35 and 45℃, respe-ctively.Linear behavior is observed betweenthe rate and the concentration of Co~(2+) ionuntil ca. 0. 15 mol·L~(-1), after the ratebecomes much faster. If no CoSO_4 ion pair catalysis exists,the rate constant of isomerization k_(obs) willhave the formk_(obs)=k_(H_2O)+k_(Na~+)[Na~+] + k_(H~+) [H~+] ++ k_(Co~(2+)[Co~(2+)]= k_1+ k_(Co~(2+)[Co~(2+)]and a linear relation between(k_(obs)- k_1)and[Co~(2+)] should be observed over the entirerange of Co~(2+) ion concentration. Failure ofthis observation indicates that a term k_(CoSO_4)[CoSO_4] should be added to the k_(obs)equation, thus the following equation shouldhold(k_(ObS)-k_1)/ [Co~(2+)] = k_(Co~(2+) + k_(CoSO_4)k_(AI) [SO_4~(2-)]in which k_(IA) stands for the constant of ionassociation of CoSO_4. By our experimentaldata,a plot of (k_(obs)-k_1/ [Co~(2+)] vs. [SO_4~(2-)]is indeed linear except for the highest[SO_4~(2-)]values where large errors arisebecause of small values of (k_(obs)-k_1) and[Co~(2+)]. From the intercept and slope ofthe line, the catalytic rate constants can becalculated. The catalytic rate constants thus obtainedat 25℃ are k_(Co~(2+))=2.13×10~(-2) (mol·L~(-1)·s)~(-1),k_(CoSO_4)=5.77×10~(-3) (mol·L~(-1)·s)~(-1) and theactivation paramaters obtained for Co~(2+) andCoSO_4 are ΔS≠=-26.4 and -96, 2 J·K~(-1).mol~(-1), ΔH≠= 74. 9 and 56. 9 kJ·mol~(-l), ΔE≠_(293)=77.4 and 59.0 kJ mol~(-1), respectively,from which the ring-opening mechanism ofthe reaction is reconfirmed.用硫酸钴催化反式-二水·二草酸根络铬(Ⅲ)的顺反异构化反应,当离子强度为1.5mol·L~(-1)、pH为3及温度分别为25、35和45℃时,发现Co~(2+)浓度不大时和反应速度成直线关系。Co~(2+)浓度大于0.15mol·L~(-1)时,25℃的反应速度大于直线关系。表明Co~(2+)和CoSO_4离子对,同时具有催化作用。测得25℃时k_(Co~(2+))=2.13×10~(-2)(mol·L~(-1)·s)~(-1),k_(CoSO_4)=5.77×10~(-3)(mol·L~(-1)·s)~(-1),并测得Co~(2+)和COSO_4的活化参数分别为 △S?=-26.4和-96.2J·K~(-1)·mol~(-1),△H?=74.9和 56.9kJ·mol~(-1)。△E?_(298)=77.4和59.4kJ·mol~(-1)。根据活化参数推论该反应为开环机理。 In this paper,the polyphenylacetylene(PPA)is synthesised by the poly-merization of phenylacetylene with two kinds of catalysts,anhydsous AlCl_3and(Ph_3P)_2PdCl_2.Using anhydrous AlCl_3 as catalyst,the cis-trans PPA isobtained.And using(Ph_3P)_2PdCl_2,the trans-cis PPA is found.Therefore,thepolymerization of phenylacetylene may follow complex catalysis mechanismwhen anhydrous AlCl_3 is used as a catalyst and it may follow metathesiscatalysis mechanism when(Ph_3P)_2PdCl_2 is used. 本文用无水AlCl_3和(Ph_3P)_2PdCl_2二种催化剂催化苯乙炔聚合,得到共轭聚合物聚苯乙炔(PPA)。对PPA 的微结构分析表明:用无水AlCl_3催化苯乙炔,得到顺-反式PPA;用(Ph_3P)_2PdCl_2催化苯乙炔得到反-顺式PPA.因此,作者认为用无水AlCl_3催化苯乙炔聚合时可能按络合催化机理反应;用(Ph_3P)_2PdCl_2催化苯乙炔聚合时可能按易位催化机理反应。&nbsp&&&&&相关查询
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Enhancer Choice in Cis and in Trans in Drosophila melanogaster
作者：James R. Morris, Dmitri A. Petrov, Anne M. Lee and Chao-ting Wu&&&&作者单位：Department of Genetics, Harvard Medical School, Boston, Massachusetts 021151 Corresponding author: Center for Genomics Research, Room 21 Harvard University, 7 Divinity Ave., Cambridge, MA 02138.
Eukaryotic enhancers act over very long distances, yet still show remarkable specificity for their own promoter. To better understand mechanisms underlying this enhancer-promoter specificity, we used transvection to analyze enhancer choice between two promoters, one located in cis to the enhancer and the other in trans to the enhancer, at the yellow gene of Drosophila melanogaster. Previously, we demonstrated that enhancers at yellow prefer to act on the cis -linked promoter, but that mutation of core promoter elements in the cis -linked promoter releases enhancers to act in trans. Here, we address the mechanism by which these elements affect enhancer choice. We consider and explicitly test three models that are based on promoter competency, promoter pairing, and promoter identity. Through targeted gene replacement of the endogenous yellow gene, we show that competency of the cis -linked promoter is a key parameter in the cis-trans choice of an enhancer. In fact, complete replacement of the yellow promoter with both TATA-containing and TATA-less heterologous promoters maintains enhancer action in cis.
【关键词】& Enhancer Drosophila melanogaster
EUKARYOTIC enhancers are able to act over long distances, sometimes interacting with promoters hundreds of kilobase pairs away. At the same time, enhancers can show a high degree of specificity, finding and interacting with their own promoter but not with other nontarget promoters. One mechanism for how this specificity is achieved suggests that intrinsic properties of enhancers and promoters limit an enhancer to a particular promoter ( L I and N OLL 1994; H ANSEN and T JIAN 1995; K APOUN and K AUFMAN 1995; M ERLI et al. 1996; O HTSUKI et al. 1998; S HARPE et al. 1998; B UTLER and K ADONAGA 2001; C AI et al. 2001; C ONTE et al. 2002 ). Here, we explore this mechanism using the phenomenon of transvection.
Transvection is a process by which a gene can affect the expression of its homologous gene on a separate chromosome in a pairing-dependent manner. It has been observed in Drosophila, where homologous chromosomes are aligned and paired in somatic cells. In addition, transvection and related processes have been observed in fungi, plants, and mammals, where extensive somatic homolog pairing has not been demonstrated, suggesting that even transient pairing interactions might have long-term consequences on gene expression (reviewed in P IRROTTA 1999; W U and M ORRIS 1999; B URGESS 2002; D UNCAN 2002; K ENNISON and S OUTHWORTH 2002; most recently B EAN et al. 2004 ).
Our studies explore transvection at the yellow gene of Drosophila. The yellow gene is required for dark pigmentation of the cuticle, including the wings, body, and bristles, and is expressed under the control of tissue-specific enhancers located in the 5' upstream region and intron of the gene ( G EYER and C ORCES 1987; M ARTIN et al. 1989; W ITTKOPP et al. 2002 ). Mutations at yellow reduce pigmentation, sometimes to a fully mutant yellow color. Interestingly, some combinations of mutant yellow alleles that reduce pigmentation in the same tissues show intragenic complementation, such that flies heterozygous for two mutant alleles show nearly wild-type pigmentation ( G EYER et al. 1990; M ORRIS et al. 1999a ). This intragenic complementation depends on the somatic pairing of yellow genes ( G EYER et al. 1990; C HEN et al. 2002; S AVITSKY et al. 2003; S. O U, J. R. M ORRIS and C.- T. W U, unpublished results). One mechanism of transvection at yellow involves the action of the wing and body enhancers of one gene in trans on the promoter of its homolog on a separate chromosome ( G EYER et al. 1990; M ORRIS et al. 1998; C HEN et al. 2002 ). As these enhancers usually prefer to act on their own promoter in cis, that is, show cis preference ( G EYER et al. 1990; M ORRIS et al. 1999a, b ), transvection at yellow provides a system where enhancer choice is changed from a state of cis preference to one that also includes trans action.
The goal of our studies is to clarify how an enhancer chooses between a cis -linked promoter and one located in trans. It has been shown that enhancer choice can be modulated by the state of the cis -linked promoter ( G EYER et al. 1990; M ART & NEZ -L ABORDA et al. 1992; H ENDRICKSON and S AKONJU 1995; C ASARES et al. 1997; S IPOS et al. 1998; M ORRIS et al. 1999a, b ). Previously, we found that mutations introduced by targeted gene replacement ( G LOOR et al. 1991; K EELER et al. 1996 ) of the yellow TATA box or initiator (Inr), two core promoter elements (reviewed in S MALE and K ADONAGA 2003 ), released the upstream wing and body enhancers to act in trans ( M ORRIS et al. 1999b ). This model can be illustrated by contrasting the abilities of two alleles, y 1 and y tata-1, to contribute enhancer activity in trans to y 82f29, which lacks both the wing and the body enhancers ( M ORRIS et al. 1998 ). As shown in Figure 1, the wing and body enhancers of y 1, which carries an intact promoter, are restricted to cis action while those of y tata-1, in which the TATA box has been altered, can act in trans as well as in cis ( M ORRIS et al. 1999b ).
F IGURE 1.- Enhancer choice in cis and in trans. (A) Cis preference of enhancers. The wing and body enhancers of y 1, with an intact promoter ( G EYER et al. 1990 ), are restricted to cis action and do not activate the promoter of y 82f29, which is a deletion of the wing and body enhancers ( M ORRIS et al. 1998 ). (B) Enhancer action in trans. In contrast, the wing and body enhancers of y tata-1, which has a 6-bp mutation in the TATA box ( M ORRIS et al. 1999b ), are able to act in trans on the promoter of y 82f29 as well as in cis on their own promoter. Both y 1 and y tata-1 have a mutated translation initiation codon to eliminate the contribution of pigmentation from enhancer action in cis. An asterisk indicates that evidence for cis action of the y tata-1 enhancers is inferred from analysis of other genotypes ( M ORRIS et al. 1999b ). The yellow gene is drawn approximately to scale and spans 7.8 kbp. W, B, Br, T,
P, solid rectangle, exon.
These observations suggest three broad classes of models for enhancer choice in cis and in trans. One proposes that enhancer choice is sensitive to some aspect of competency involving the cis -linked promoter, such as transcription rate, the binding of specific transcription factors to the promoter, or a particular chromatin state. This model is in line with studies of enhancers and promoters in cis -linked configurations ( S HARPE et al. 1998; C AI et al. 2001; C ONTE et al. 2002 ). Another model proposes that enhancer choice is influenced by the effect of promoter mutations on the state of pairing between two yellow alleles. That is, sequence heterology resulting from mutations in the cis -linked promoter or flanking regions may lead to promoter unpairing, and this unpairing may then render the intact promoter in trans to an enhancer more attractive, perhaps through conformational changes or by making it more accessible to transcription factors ( S IPOS et al. 1998; M ORRIS et al. 1999a ). A third model suggests that mutations in core promoter elements, by altering key promoter sequences, exert their effects on enhancer action by changing the identity or signature of the promoter. Such changes might cause an enhancer to seek a native promoter on a separate chromosome rather than interact with a cis -linked promoter that is of the wrong identity. Studies of cis -linked enhancers and promoters have revealed that enhancers can discriminate among different promoters on the basis of the presence or absence of core promoter motifs ( O HTSUKI et al. 1998; B UTLER and K ADONAGA 2001; C ONTE et al. 2002 ).
Here, we use targeted gene replacement at yellow to further examine these three models. In particular, we ask whether a mutation of a yellow core promoter element, the TATA box, releases enhancers to act in trans due to its effects on promoter competency, pairing, or identity. Surprisingly, we find that while a 6-bp mutation of a core promoter element releases enhancers, complete replacement of 193 bp of the yellow promoter with any of three heterologous promoters fails to release enhancers to trans action. We argue that the roles of promoter pairing and identity in the cis-trans decision are likely to be at most secondary to that of promoter competency. In addition, we consider our data in light of the possibility that competency of a promoter may reflect features of the promoter in addition to transcription strength.
MATERIALS AND METHODS
Scoring of pigmentation:
Pigmentation was scored as previously described ( M ORRIS et al. 1998 ). We scored 1- to 3-day-old females using a five-point scale, where 1 represents the null or nearly null state and 5 represents the wild-type or nearly wild-type state. For consistency, flies were scored by comparison to flies whose wing and body pigmentation was previously reported, including y 1 / y 1 (1, 1), y 2 / y 2 (1, 1-2), y tata-1 / y 82f29 (2, 1-2), y tata / y tata (3, 2), y 1#8 / y 82f29 (3, 3), y 1#8 / y 2 (4, 4), and y + / y + (5, 5). However, because pigmentation represents a continuum, there is some variation of pigmentation for flies scored at the same value. Complementation between two alleles required that wing or body pigmentation was one point darker on the pigmentation scale than that of corresponding tissues in females homozygous for either allele. Flies were cultured at 25& & 1& as previously described ( M ORRIS et al. 1998 ). Crosses were done with three to four females mated to three or more males in vials that were brooded daily. Both temperature and crowding were carefully controlled.
Plasmid construction:
The templates used in the targeted gene replacement experiments were full-length 7.8-kbp yellow genes (GenBank accession nos. X06481 and X04427 ) with the designated sequence changes (Figure 2) cloned into a modified Bluescript plasmid (Stratagene, La Jolla, CA) in which the Kpn I site is replaced by an Xba I site (pBSX). pUC8ySB is a pUC8 plasmid containing the wild-type 3.1-kbp Sal I- Bam HI pUC8y1SB contains the A-to-C mutation of y 1; pUC8ySBtata contains the 6-bp sequence change in the TATA b pUC8ySBtata-1 contains the 6-bp TATA mutation and the A-to-C mutation of y 1; pBSXyBG is pBSX containing the 4.7-kbp Bam HI- Bgl II yellow fragment ( M ORRIS et al. 1999b ). All sequence changes were first incorporated into pUC8ySB or one of its derivatives noted above, which was then digested with Sal I and Bam HI and cloned into pBSXyBG to generate full-length templates in pBSX. All changes were confirmed by sequencing.
The four insertional promoter templates, tT, tT-1, TT, and TT-1, were made by introducing a double-stranded linker (see below) at the Eag I site of pUC8ySB, pUC8y1SB, pUC8ySBtata, or pUC8ySBtata-1, respectively.
The eight promoter scan (ps) templates were made using a PCR strategy with one of the two primers carrying the desired sequence changes. For ps1 and ps1-1, primers 1 and m13rev (primer sequences shown below) were used to generate PCR products with pUC8ySB or pUC8y1SB as a template, respectively. The resulting PCR products were digested with Eag I and Bam HI and cloned into pUC8ySB. For ps2 and ps2-1, primers 2 and m13rev were used in a similar strategy. For ps3 and ps3-1, primers 3 and 4 were used with pUC8ySB as a template, and the resulting PCR products were digested with Kpn I and Eag I and cloned into pUC8ySB or pUC8y1SB, respectively. For ps4 and ps4-1, primers 3 and 5 were used in a similar strategy.
The six heterologous promoter templates were made by first cloning the Kpn I- Bam HI yellow fragment from pUC8ySBps4 into pBS to make pBSyKBps4. Primer pairs 6/7, 8/9, and 10/11 were then used to PCR amplify a 193-bp fragment from the even skipped ( eve ), heat-shock protein 70 ( hsp70 ), and white ( w ) promoter regions of wild-type Canton-S genomic DNA, respectively. These were TA cloned (Invitrogen, San Diego), confirmed by sequence analysis, cut with Cla I and Afl II, and cloned into pBSyKBps4. The resulting plasmid was digested with Kpn I and Bam HI and cloned into pUC8ySB.
Constructs for P -element-mediated transformation were made by digesting the three pBSX plasmids containing the heterologous promoters with Hin dIII and Not I, religating the resulting 2.9-kbp pBSX fragment and the 5.5-kbp 3' yellow fragment, digesting the resulting plasmid with Xba I, and cloning this fragment into the Xba I site of pCaSper3.
Targeted gene replacement and P -element-mediated germ-line transformation:
Targeted gene replacement was carried out as previously described using y h12w+ as a target allele on a chromosome that also carried w 1118 and plasmid sources of templates and transposase at concentrations of 1.0 and 0.25 mg/ml, respectively ( K EELER et al. 1996; M ORRIS et al. 1999b ). On average, 500 embryos were injected per construct, with a larval survival rate of 40% and a conversion rate of 1% (of total embryos injected). Candidate lines were screened by single-fly PCR using primers 12 and 13, which give an 874-bp PCR product. Those lines giving wild-type-sized products were confirmed by digesting the PCR product with restriction enzymes diagnostic of the desired changes and sequence analysis. Southern analysis of genomic DNA digested separately with Hin dIII/ Bam HI and Pst I and hybridized to a full-length yellow probe was performed to confirm the integrity of the yellow gene (data not shown). P -element-mediated transformation was done as previously described ( R UBIN and S PRADLING 1982; M ORRIS et al. 1998 ) using 0.5 mg/ml construct, 0.1 mg/ml helper plasmid, and the Df(1)y - ac - w 1118 stock as the host.
Northern analysis:
Flies were allowed to lay eggs for 24 hr in bottles at 25& and pupae were collected 8 days later. Total RNA was collected [Eppendorf (Madison, WI) Eukaryotic Perfect RNA] and poly(A) + RNA was isolated [Promega (Madison, WI) PolyATtract] and electrophoresed (5 &g/lane) on a 1% agarose/formaldehyde gel followed by hybridization with a 32 P-labeled Eco RI- Bgl II yellow fragment from the second exon and rp49 as a loading control. Quantitation was based on band intensity relative to the average of the band intensities for the four data points from two independent y + lines generated by targeted gene replacement. The average level of steady-state mRNA in our wild-type Canton-S line was 91% (over three trials) of the level in our y + control lines (data not shown). Preliminary experiments using Canton-S pupae collected daily from days 6-10 indicated that day 8 showed maximal steady-state yellow mRNA expression (data not shown). On the basis of these results, day 8 pupae were used in subsequent experiments. However, because time courses were not done for each of the alleles, it is possible that maximum expression is shifted earlier or later compared to that of Canton-S pupae.
Primers and linkers:
Primer and linker sequences used in this study are indicated below in 5' to 3' orientation with mutated nucleotides in boldface type.
AAAACGCGGCCG GGTACC TATGGCCACCAGTCGTTACCGCGCCACGGTCCACAGAAG;
AAAACGCGGCCGACATAT AGATCT CACCAGTCGTTACCGCGCCACGGTCCACAGAAG;
GCAGTCGCCGATAAAGATGAACACAG;
ATATGTCGGCCGCGTTTTATATGAAGGTTTTTTTCTCC ACTAGT GAAGACAGGCCAATGAAAATGAAAACG;
ATATGTCGGCCGCGTTTTATATGAAGGTTTTTTTCTCCGAAGACGAAGACAGG ATCGAT AAAATGAAAACGAAGGCG;
ATCGATGACGGCGGCCATTTGCCTGCAGAGCGCAGCGGTATAAA;
CTTAAGGGCTCTCCAGGTTGTAGGTTCGGTATCCGTGAATGTTT;
ATCGATCGCCTCGAATGTTCGCGAAAAGAG;
CTTAAGCTGGTTACTTTTAATTGATTCACT;
ATCGATCGCTGCGTCCGCTATCTCTTTCGCCACC;
CTTAAGTCACCACCCCAATCACTCAAAAAACAAA;
GAGCCTCCTGGCCTTACAATTTAC;
ATTTAACTTCCACTTACCATCACGCC;
Linker: 5'-GGCCAAAAAAACCTTCATATAAAACGC-3'
3'-TTTTTTTGGAAGTATATTTTGCGCCGG-5'.
Testing the role of transcription in the control of enhancer choice:
Previously, we demonstrated that a 6-bp mutation of the TATA box ( y tata ), the initiator ( y inr ), or the TATA box and initiator ( y tata-inr ) is sufficient to support transvection by releasing the wing and body enhancers to act in trans ( M ORRIS et al. 1999b; Table 1; Figure 2 ). Because these mutations were made in core promoter elements, the data suggested a model in which disruption of promoter competency, in particular its ability to support transcription, releases enhancers to act in trans. Here, we tested this model by determining the steady-state level of yellow expression through Northern analyses. We compared the level of yellow mRNA in flies carrying y tata, y inr, or y tata-inr to that in control flies carrying a wild-type y + gene. Our y + control flies were generated by the protocol used to generate y tata, y inr, and y tata-inr, that is, through targeted gene replacement ( G LOOR et al. 1991; K EELER et al. 1996 ) of the mutant yellow allele in our standard starting line, y h12w+ w 1118 (see MATERIALS AND METHODS; the w 1118 null mutation of the white gene is present in all our convertant lines, but is not mentioned further). We found that steady-state levels of yellow mRNA are reduced in y tata, y inr, and y tata-inr to 1, 3, and 1%, respectively, of the level found in control y + flies ( Figure 3 ). These data are consistent with a model in which promoters that have been transcriptionally compromised release their enhancers to act in trans.
TABLE 1 Complementation data
F IGURE 2.- Partial sequences of wild-type and mutant yellow alleles generated by targeted gene replacement. Underlined bases, heat-shock element, TATA box, and I bases in boldface type, asterisk, transcrip //, break in sequence. For yellow, // indicates TTACCGCGCCACGGTCCACAGAAGAGGATTAAAAAAATATCACACAGCCGAAGGCTAGAGAAGAACCCCCTTAGCTGAACATATATAAACAAATATATTTTTTTTTATTGCCAACACACT. For eve, // indicates CGAGCTGTGACCGCCGCACAGTCAACAACTAACTGCCTTCGTTAATATCCTCTGAATAAGCCAACTTTGAATCACAAGACGCATACCAAACATTCACGGATACCGAACCTACAACCTGGAG. For hsp70, // indicates CAATTCAAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGCAGTAAAGTGCAAGTTAAAGTGAATCAATTAAGAGTAA. For w, // indicates CACTTTGTCAGCGGTTTCGTGACGAAGCTCCAAGCGGTTTACGCCATCAATTAAACACAAAGTGCTGTGCCAAAACTCCTCTCGCTTCTTATTTTTGTTTGTTTTTTGAGTGATTGGGGTG.
F IGURE 3.- Quantitation of transcript levels following Northern analysis of yellow alleles. Transcript levels from the y + control lines were averaged and set at 100%, and transcript levels for other alleles are expressed as a percentage of this average. Quantitation was based on band intensity. Height of bars (indicated by number over bar) represents the mean of two or more experiments. Error bars indicate one standard error on either side of the mean.
This interpretation further predicts that restoration of transcription to a transcriptionally compromised allele will recapture the upstream enhancers and prevent transvection. We tested this prediction by inserting a wild-type TATA box downstream of the mutated TATA box in y tata ( Figure 2 ) and then testing the resulting allele for transcription and the ability of its enhancers to act in trans. Note that we chose to restore transcription to y tata instead of y inr because y tata is the more transcriptionally compromised of the two ( Figure 3 ) and therefore provides a stronger test. Specifically, we used targeted gene replacement to insert the 27-bp region extending from position -44 to -18 of the wild-type yellow gene into the y tata allele at position -17/-18 (nucleotides numbered relative to the transcription start site of +1). The resulting allele carried a wild-type promoter 3' of position -44. We called the new allele y tT, where t and T represent the mutated and wild-type TATA boxes, respectively, and their order represents their 5' to 3' order in vivo. Homozygous y tT / y tT and hemizygous y tT / Df (where Df represents the y - ac - w 1118 chromosome that is deficient for
M ORRIS et al. 1998 ) flies showed fully wild-type pigmentation in all cuticular structures, including the wings, body, and bristles, similar to our y + control flies ( Table 1 ). However, by Northern analysis, steady-state yellow mRNA levels were only 11% of the level seen in y + control flies ( Figure 3 ).
Next we determined the ability of the wing and body enhancers of y tT to act in trans. To this end, we first generated a companion protein-null derivative of y tT such that translation of the transcripts made in the presence of the promoter alterations would not obscure our tests of the ability of the upstream enhancers to act on a promoter in trans. As was done for all subsequent companion alleles mentioned below, this protein-null derivative was generated by targeted gene replacement and bore, in addition to the changes in the promoter region, an A-to-C change in the ATG translation initiation codon. This change is identical to that found in the y 1 protein-null allele ( G EYER et al. 1990 ) and therefore, as expected, the companion allele for y tT, called y tT-1, gives a fully mutant phenotype ( Table 1 ). We then determined whether y tT-1 could release its wing and body enhancers to act in trans by asking whether it complements y 82f29 ( Figure 1 ) and three other alleles, y 2, y 62a, and y 2374, which, like y 82f29, lack strong wing and/or body enhancer activity ( M ORRIS et al. 1999b ). We found that y tT-1 does not complement each of these tester alleles. This finding suggests that restoration of transcriptional competency, as revealed by increased pigmentation and transcript levels in y tT compared to those of y tata, is sufficient to recapture the upstream enhancers and prevent transvection. Interestingly, yellow transcript levels in y tT flies reach only to the 11% level compared to the y + control ( Figure 3 ), indicating that transcription need not be restored to wild-type levels for the upstream enhancers to be restricted to cis action.
These data also argue against a model in which local promoter unpairing as a result of structural heterozygosity between two alleles in the promoter region is sufficient to release enhancers to trans action. Specifically, the 27-bp insertion present in y tT would be predicted to unpair the promoter region when paired with a tester allele, yet it does not support transvection. To test this interpretation, we made a second insertional yellow allele, called y TT, that differs from y tT only in that it carries a wild-type TATA box in place of the mutated TATA box ( Figure 2 ). The y TT allele produced wild-type pigmentation in homozygous and hemizygous flies, and its protein-null companion allele, y TT-1, failed to complement each of the four tester alleles ( Table 1 ). These findings provide additional support for the interpretation that local promoter unpairing as a result of sequence heterology between two alleles is not sufficient to release enhancers to act in trans.
Not all sequence changes in the promoter release enhancer to trans action:
These results indicate a strong correlation between promoter elements involved in transcription and those involved in enhancer choice. We next decided to test the strength of this correlation by introducing mutations in noncore promoter sequences. Will all elements that affect transcription also affect enhancer choice and vice versa, or are there elements dedicated to one process but not the other? Using targeted gene replacement, we made four ps mutations, y ps1, y ps2, y ps3, and y ps4 ( Figure 2 ), each of which carried a 6-bp substitution in the promoter region, and four corresponding protein-null companion alleles, y ps1-1, y ps2-1, y ps3-1, and y ps4-1. Our design of these alleles was guided by the presence within the yellow promoter of an 80-bp region, extending from position -43 to +37, that shows 91% sequence identity with the homologous promoter region of the yellow gene of Drosophila subobscura, a species that diverged from D. melanogaster 30 million years ago ( M UNT & et al. 1997 ). This sequence identity suggests that there might be important functional elements in this region, a prediction supported by sequence comparisons with other Drosophila species (M UNT & et al. ; W ITTKOPP et al. 2002 ). With this in mind, we placed two mutations ( y ps1 and y ps2 ) within and two ( y ps3 and y ps4 ) outside this region of conservation.
In spite of the different placements relative to the conserved region, however, all four mutations have similar pigmentation and transcriptional phenotypes. Homozygous or hemizygous y ps flies show wild-type pigmentation ( Table 1 ) and steady-state yellow mRNA levels in these mutants are between 16% ( y ps1 ) and 35% ( y ps3 ) of levels seen in our y + control flies ( Figure 3 ). The finding that mutations in noncore promoter elements nevertheless have a strong negative effect on steady-state mRNA levels is consistent with mutational analyses of noncore elements in other systems (for example, D UDLEY et al. 1999; W RAY et al. 2003 ).
None of the companion alleles supported transvection when placed in trans to the four tester alleles ( Table 1 ). These data suggest that not any 6-bp mutation in the promoter region allows transvection, even though all that we tested reduce transcript levels. In light of our observation that mutations of the yellow TATA box and Inr do release enhancers ( M ORRIS et al. 1999b ), these data demonstrate the importance of core promoter elements in enhancer choice. Furthermore, as mutations in core elements reduce transcript levels more significantly than mutations in noncore sequences ( Figure 3 ), one interpretation is that only mutations that most severely compromise transcription release enhancers to trans action.
The data also suggest a second way to look at the role of promoter competency in enhancer choice. Specifically, it may be that the primary feature of the promoter that guides enhancer choice is its integrity as defined, for example, by the array of core elements rather than by an absolute rate of transcription. According to this interpretation, the ps alleles maintain cis preference of enhancers because their core promoter elements are intact. Furthermore, this model is consistent with the ability of y tT and y TT to in spite of their low steady-state transcript levels, they nevertheless have an intact configuration of core promoter elements.
Enhancer action in cis can be maintained by heterologous promoters:
The data thus far support a key role for promoter competency in the cis-trans choice of an enhancer. Here we consider the third model in which promoter identity guides enhancer choice. In particular, we asked whether the 6-bp alteration in the TATA box changes the yellow promoter from a TATA-containing to a TATA-less promoter, thereby making it a poor target for the enhancers. Specifically, will the upstream enhancers of a yellow allele with a functional but heterologous promoter in place of the native promoter choose the heterologous promoter in cis rather than the native promoter in trans ?
To answer this question, we used targeted gene replacement to substitute 193 bp of the yellow promoter from position -63 to +130, including the TATA box and Inr but not the translation initiation codon, with 193 bp of sequence from the promoter regions of the Drosophila eve, hsp70, and w genes ( Figure 2 ). The resulting alleles were called y eve, y hsp70, and y w, and their protein-null companion alleles were called y eve-1, y hsp70-1, and y w-1. The eve promoter most closely resembles that of yellow in that both have a TATA box and Inr ( M AC D ONALD et al. 1986 ). The hsp70 promoter, like that of yellow and eve, has a TATA box and Inr but, in addition, contains multiple heat-shock elements ( P ERISIC et al. 1989 ), one of which is included in the 193-bp sequence inserted into yellow. The white promoter is TATA-less, but does have an Inr and downstream promoter element sequences ( K ADONAGA 2002 ).
All three heterologous promoters gave dark pigmentation in homozygous or hemizygous flies ( Table 1 ), indicating that they can direct yellow transcription. Flies bearing the y eve allele were indistinguishable from wild-type flies, while flies bearing the y hsp70 allele were at least as dark, if not darker, than wild-type flies. In addition, y hsp70 flies had a dusky appearance with dark pigmentation even in abdominal interbands, which are the normally lightly pigmented regions between the dark abdominal bands. The y w flies were less dark than wild-type flies. Consistent with these observations, steady-state levels of yellow transcripts reached 80 and 71% of wild-type levels in y eve and y hsp70 flies, respectively, but only 12% in y w flies ( Figure 3 ).
To verify that transcription from these heterologous promoters is enhancer driven and not constitutive, we made constructs that carried promoter replacements identical to those present in y eve, y hsp70, and y w, but that were deleted for the wing and body enhancers, and determined the pigmentation levels that they direct when integrated into the genome by P -element-mediated germ-line transformation. If transcription of y eve, y hsp70, and y w is enhancer dependent, then flies bearing these enhancerless transgenes should show mutant pigmentation. If expression of y eve, y hsp70, and y w is constitutive, then the transgenic flies should instead show dark pigmentation. Flies carrying the eve and white enhancerless transgenes showed fully mutant wing and body pigmentation in all five and three, respectively, independent transgenic lines, indicating that the wing and body pigmentation seen in y eve and y w flies is in fact the result of enhancer-dependent transcription. The eight independent lines bearing the hsp70 enhancerless transgene differed from each other, showing a range of wing and body pigmentation from fully mutant to nearly wild type. This observation may reflect position effects or might suggest that the dark pigmentation seen in y hsp70 flies is partially constitutive, but that to achieve consistently full pigmentation, input from an enhancer is required. Significantly, all of the transgenic lines carried an intact bristle enhancer and showed wild-type bristle pigmentation, indicating that the transgenes are capable of yellow expression in their ectopic locations.
We then tested the ability of y eve, y hsp70, and y w to release the wing and body enhancers to act in trans by placing each of their corresponding companion alleles in trans to the four tester alleles and assaying complementation. None of the companion alleles complemented the tester alleles ( Table 1 ), suggesting that yellow enhancers prefer to act in cis, even on a foreign promoter, rather than act in trans on a wild-type yellow promoter. As these heterologous promoters maintain cis preference of the yellow enhancers in spite of their different sequences and identities relative to the native yellow promoter, our data indicate that promoter pairing and identity are unlikely to play primary roles in enhancer choice at yellow.
DISCUSSION
At the outset of our studies, we considered three mechanisms that may govern how an enhancer chooses between a cis -linked promoter and one located in trans : by some aspect of promoter competency, by pairing-mediated changes in gene topology, and by promoter identity. Although our data do not rule out any model, they argue that the latter two mechanisms do not, on their own and in their simplest form, dictate cis-trans decisions of the wing and body enhancers of yellow. In contrast, our data draw attention to the transcriptional process and the integrity of the promoter.
Significantly, consideration of the alleles from the point of view of transcription shows that those supporting transvection reduce transcript levels below 3% compared to levels in our y + control flies, while those that do not support transvection maintain transcript levels above 11% ( Figure 3 ). The difference in transcript levels between the two groups of alleles is significant (Mann-Whitney U -test, P = 0.005) and suggests that the trancriptional competency of a promoter may need to be compromised below a certain threshold, as assayed by steady-state transcript levels, before the cis -linked enhancers can be released to act in trans.
What this hypothetical transcription-based threshold represents in biological terms is not clear. It may reflect a particular rate of transcription, the binding of a specific transcription factor, the adoption of a particular chromatin state, or another aspect of promoters that can affect the attractiveness of a promoter to an enhancer ( L I and N OLL 1994; H ANSEN and T JIAN 1995; K APOUN and K AUFMAN 1995; M ERLI et al. 1996; O HTSUKI et al. 1998; S HARPE et al. 1998; B UTLER and K ADONAGA 2001; C AI et al. 2001; C ONTE et al. 2002 ). Importantly, as transvection requires many events before transcription, such as homology sensing and pairing, the cis-trans choice of a yellow enhancer may occur much earlier than transcription, possibly making a threshold defined by transcript levels a poor reporter of an earlier key step. Choice may also be a dynamic process, varying from cell to cell or tissue to tissue ( G OLIC and G OLIC 1996; G UBB et al. 1997; reviewed in W U and M ORRIS 1999; K ENNISON and S OUTHWORTH 2002 ). Indeed, we have not yet determined whether yellow enhancers can alternate from moment to moment between the cis and trans promoters, perhaps interacting simultaneously with both. Finally, as has been observed at the bithorax complex ( G OLDSBOROUGH and K ORNBERG 1996; C ASARES et al. 1997; S IPOS et al. 1998 ), it is formally possible that yellow enhancers are released to trans action at a threshold higher than we have observed, even when the cis promoter is entirely wild type and fully functional. If true for any of the noncomplementing genotypes we have tested, such interactions are nonproductive, ineffective, or too infrequent to affect pigmentation.
Interestingly, all nine alleles that maintain cis preference of enhancers also have an intact configuration of known core promoter elements. The insertional alleles have an intact configuration 3' of position -44, the promoter scan alleles do not affect core elements, and the heterologous promoter alleles have foreign but otherwise intact promoter sequences. By contrast, the three alleles that do support transvection ( y tata, y inr, and y tata-inr ) all have mutations in core promoter elements ( M ORRIS et al. 1999b ). This observation highlights the possibility that enhancer choice between two promoters may be influenced by the integrity of the promoter as determined by the configuration of core elements, the factors bound to them, and/or the chromatin state that they generate. That is, the nine alleles that maintain cis preference of their enhancers might do so because they each have an intact array of core promoter elements in spite of their differences in sequence, identity, and transcriptional profiles. This feature may be a molecular mark of a fully competent promoter in terms of transcription or, at the extreme, may reflect a promoter function independent of the transcriptional process.
A comparison of y inr with y w illustrates this point. The y inr allele has a 6-bp mutation in the initiator ( M ORRIS et al. 1999b ), while y w has a white promoter substituted for the yellow promoter ( Figure 2 ). Steady-state levels of yellow mRNA are 3% for y inr and 12% for y w as compared to that of our y + controls and, as predicted by the threshold model, y inr supports transvection while y w does not. Interestingly, however, y w flies show lighter wing and body pigmentation as compared to y inr flies ( Table 1 ) in spite of their higher transcript levels ( Figure 3 ). It may be that white sequences in the 5'-untranslated region lead to the production of unstable transcripts, so that while transcript levels are relatively high, protein expression is not. If so, the transcription-based threshold would still apply. However, it may also be that yellow expression in tissues other than wing and body accounts for the discrepancy between phenotype and transcript levels of y w. If so, the transcription-based threshold would not apply in this case. Instead, it may be that the enhancers of y inr are released to act in trans because y inr carries a mutated core promoter element and therefore they perceive their promoter as inadequate, while the enhancers of y w are not released because they perceive the geography of their promoter as intact.
The ability of y w as well as y eve and y hsp70 to maintain cis preference also indicates that promoter pairing and identity are unlikely to be key determinants of cis-trans choice. Interestingly, at Ultrabithorax ( Ubx ), regulatory regions can act both in cis with a heterologous P -element promoter and in trans on their own promoter ( C ASARES et al. 1997 ). This observation could reflect differences between yellow and Ubx in terms of the involvement of transposable elements, participating enhancers and promoters, or mechanisms of transvection.
In sum, our data suggest that the cis-trans choice of the yellow wing and body enhancers rests to a significant degree on the competency of the cis -linked promoter. Furthermore, our data call attention to two aspects of promoter competency with respect to the cis-trans choice: transcriptional competency and intactness of the promoter. A model of enhancer choice based on promoter function and integrity provides an explanation of why enhancers are able to detect small changes in key promoter elements but are apparently insensitive to complete replacement of the promoter.
ACKNOWLEDGEMENTS
The authors especially thank P. Geyer for years of close collaboration and insightful discussions, G. Gloor for invaluable advice on targeted gene replacement, J. Bateman, W. Bender, R. Emmons, C. Kaplan, J. Lokere, S. Ou, B. Williams, and F. Winston for discussions, and A. Moran for generous technical assistance. This work was supported by a National Institutes of Health grant (RO1 GM61936) to C.-t.W. and support from the William F. Milton Fund and the Harvard University Society of Fellows to J.R.M. and D.A.P.
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