IOMMENT
Insistent and intransigent:a phage Mu enhancer functions in trans MARTIN R. BOOCOCK, SALLY-J. ROWLAND, W. MARSHALLSTARK AND DAVID J. SHERRAIT INSTITUTEOF GENETICS,UNIVERSITYOF GLASGOW, GLASGOW,UK Gll 5JS. In the intricate regulatory games that control gene expression, two major classes of players can be distinguished. On the one hand are cis-acting elements in the DNA and RNA sequences, such as promoters, enhancers and splice sites. To be worthy of the name, cis-acting elements are expected to interact exclusively with other genetic elements in the same DNA or RNA chain. The c/~-acting sequences are recognized and acted upon by a menagerie of freely diffusible transacting factors, including sequencespecific binding proteins, intermediary factors, and the protein and nucleoprotein machinery of transcription and RNA processing 1. In cis and in trans Why do ci~acting elements not cause regulatory chaos by interacting with DNA sequences in trans? Are they functionally incapable of unruly behaviour, or are they held in check by restrictions on access between different DNA segments? It has been suggested that a continuous DNA path between two cis-acting elements is essential for
their proper communication. However, in a number of systems where this has been tested directly, it turns out that a continuous DNA linkage is dispensable, although some elements do need to be tethered in close proximity to interact successfully 2~8. These results dealt a heavy blow to the idea that enhancers function remotely from their targets, acting either as binding sites for regulatory topoisomerases, or a& entry sites for factors that slide or assemble continuously along the DNA towards the target2, 8. In recently published work, Surette and Chaconas9 have gone a step further. They show that an enhancer element required for transposition in bacteriophage Mu can function efficiently in trans, and that it does not have to be tethered to its two target sites. The enhancer intervenes transiently in the assembly of a nucleoprotein complex between the two ends of the Mu genome, and is not involved in any of the subsequent catalytic steps of the transposition reaction (Fig. 1).
Synapsis and looping When bacteriophage Mu enters its lyric cycle, the 37 kbp proviral genome undergoes multiple rounds of replicative transposition within the chromosomal DNA of the bacterial host. Three c/s-acting elements participate in the transposition reaction: the left and right ends of the Mu genome, and an enhancer element normally located 950 bp from the left end of Mu. In addition, four proteins - MuA, MuB, IHF and HU - are required for transpositional recombination in vitro a°,l~. In the earliest detectable transposition intermediate, the type 0 complex, the MuA protein synapses the ends of the Mu genome, dividing the circular DNA into two supercoiled loops (Fig. 1). The DNA strands are then nicked to expose free 3' hydroxyl groups at the extreme left and right ends of the Mu sequences, giving a stable type I complex with one relaxed DNA loop and one supercoiled loop. The subsequent steps, in which the Mu sequences are 'pasted' into the target DNA, are interesting 12, but do not concern us here.
0 O L
R
strand ~OH R
Ip
cleavage L
R
•
Mg2+
strand transfer reaction
J
t a r g e t DNA
ancer
MuB E
substrate + MuA
hypothetical 3-site synapse
type 0 complex
E
type I complex
FIGg A simplified view of the early steps in phage Mu transposition. Arrows (L, R and E) represent the left and right Mu ends and the enhancer; shaded areas represent protein. MuA binds to multiple sites in the Mu ends and two sites in the enhancer; IHF binds to one site in the enhancer. The linking DNA sequences are not shown to scale. l~G ~AAV1992 VOL. 8 NO. 5 ©1¢~)2 Else',ier Science Publisher~, Lid (I!K)
[~]'OMMENT
•
B : ' i n a c t i "v e
C : i n a c t i v" e
FIGM
Active and inactive configurations of the Mu transposition enhancer nJ3J4. (A) Enhancer in standard configuration; (B) enhancer inverted (very low activity); (C) enhancer outside the Mu ends (inactive). For description of arrows and shaded areas, see Fig. 1. The formation of stable transposition intermediates normally requires an enhancer in cis 11,13'14, although the enhancer has not been found associated with the ends of the Mu g e n o m e in any intermediates yet characterized. The enhancer consists of an IHFbinding site w e d g e d between two binding sites for MuA. IHF is required for the enhancer to function at physiological supercoil densities, and is believed to promote interactions between the adjacent MuA sites by bending the DNA 11. Different domains of the MuA protein recognize the different sequence motifs found in the enhancer and in the ends of the Mu genome, raising the interesting possibility that individual molecules of MuA could synapse sites in the enhancer and in the Mu ends 13A4. Surette and Chaconas have now shown that the enhancer functions efficiently w h e n supplied in trans, on a 199 bp DNA fragment9. The enhancer is required only for assembly of the type 0 complex, since it can be removed by electrophoresis before formation of the type I complex.
twined configuration (Fig. 1). This specific configuration is suggested to be the basis for site selectivity in the context of a supercoiled DNA substrate. In agreement with this model, the enhancer fails to work in cis w h e n its orientation is inverted (Fig. 2B), or when it is located 'outside' the two Mu ends (Fig. 2C) 11,13,14, Surette and Chaconas argue that the local configuration postulated for the three-site synapse should be more readily accessible if the enhancer is supplied in trans (Fig. 3A). The impressive activity of the unlinked
A: Mu transposition
B: Mu Gin recombination
Selection rules The enhancer is implicated in selecting left and right Mu ends in cis in the correct head-to-head orientation3, TM. The enhancer is thought to participate in a sl'rortlived three-site synapse that precedes the type 0 complex, with the three sites adopting a specific inter-
enhancer in their experiments provides further support for the proposed intertwined three-site synapse. In trans, the enhancer works best on a short linear DNA fragment, and is less proficient when embedded in a larger linear or circular DNA; this may be due to conformational difficulties in inserting the enhancer into the synapse9. An enhancer has been implicated in the assembly of a catalytically active nucleoprotein complex in two other systems: FISdependent DNA inversion by Mu Gin, and NtrC regulation of glnA transcription6,15. These systems differ in that the enhancer functions in trans only when tethered to the target by catenation of two DNA circles (Fig. 3). For the Mu sis enhancer, simple catenation is not sufficient; the circles must be multiply entwined and supercoiled (Fig. 3B) 16, indicating that DNA topology is all-important in assembly of the three-site synapse 17. Do enhancers work well in cis simply because they are tethered close to the target site? The Mu transposition enhancer functions optimally in trans at a concentration of approximately 100 nM, but is still not as effective as an enhancer in cis 9. For comparison, DNA segments separated by 1 kbp or 10 kbp in a large nonsupercoiled circle interact at effective
C: NtrC
FIGIFI
Different degrees of tethering are required for different enhancers to function in trans. (A) Mu transposition enhancer (E) on unlinked linear DNA interacts with Mu ends (L, R) on a supercoiled substrate9 (as in Fig. 1). (B) In a supereoiled, multiply interlinked catenane, the sis enhancer stimulates recombination between the two gix sitesl6; sis and gix contain binding sites for FIS and the Gin protein of Mu, respectively. (C) In a simple catenane, the E. coli glnA enhancer stimulates the formation of an open transcription complex at the glnA promoter. NtrC protein binds at multiple sites in the enhancer; RNA polymerase with the ~54 subunit binds to the promoter to give a closed complex (,. "HG MAY1992 VOL. 8 NO. 5
I'OMMENT concentrations of approximately 60 nM and 3 nM, respectively (effective concentrations may be higher in a supercoiled molecule) TM. Hence, it is not entirely surprising that a high concentration of enhancer is required in trans., it may indicate that the interaction with the target is not controlled by single-hit kinetics, but is weak and reversible. If an enhancer DNA-protein complex can activate its target in trans, w h y is the activator protein alone not sufficient? In Mu transposition, it is evident that the MuA protomers must be delivered to the target sites in a very precise configuration that depends on IHFinduced bending of the enhancer sequences9,n. Transcriptional activator proteins from eukaryotes are generally inactive or inhibitory in isolation from their specific binding sites in DNAI; we do not yet know how they are presented to their target proteins. Do gene regulatory sequences necessarily interact in cis in the cell nucleus? In Drosophila, interactions between regulatory sequences on homologous chromosomes have been well documented for a num-
~STUDENTS
-
ber of genes, although these 'transvection' effects are not commonplace 19. Transvection at several loci requires the zeste gene product, which is believed to synapse distant chromosomal sites, bringing other regulatory sequences into closer proximity 2°. For most genes, however, a defective enhancer cannot be complemented by a functional enhancer supplied in trans on a homologOus chromosome, or 500 kbp away on the same chromosome. To understand why, we need to learn more about chromatin dynamics in the vicinity of promoters and enhancers2E
References 1 Ptashne, M. (1988) Nature 335, 683--689 2 Mt~ller, H-P~, Sogo, J.M. and Schaffner, W. (1989) Cell 58, 767-777 3 Craigie, R, and Mizuuchi, K. (1986) Cell 45, 793-800 4 Stark, W.M., Sherratt, D.J. and Boocock, M.R. (1989) Cell 58, 779--790 5 Dunaway, M. and Droge, P. (1989) Nature 341,657-659 6 Wedel, A. et al. (1990) Science 248, 486--489
SUBSCRIBE
TO
TIG
7 Rothberg, I., Hotaling, E. and Sofer, W. (1991) Nucleic Acids-Res. 19, 5713--5717 8 Plon, S.E. and Wang, J.C. (1986) Cell 45, 575-580 9 Surette, M.G. and Chaconas, G. (1992) Cell68, 1101-1108 10 Mizuuchi, K. (1983) Cell35, 785-794 11 Surette, M.G., Lavoie, B.D. and Chaconas, G. (1989) EMBOJ. 8, 3483--3489 12 Mizuuchi, K. and Adzuma, K. (1991) Cell 66, 129-140 13 Leung, P.C., Teplow, D.B. and Harshey, R.M. (1989) Nature 338, 656458 14 Mizuuchi, M. and Mizuuchi, K. (1989) Cell 58, 399~i08 15 Kanaar, R. et al. (1990) Cell 62, 353-366 16 Kanaar, R., van de Putte, P. and Cozzarelli, N.R. (1989) Cell 58, 147-149 17 Heichman, K.A. and Johnson, R.C. (1990) Science 249, 511-517 18 Wang, J.C. and Giaver, G.N. (1988) Science 240, 300-304 I 9 Pirrotta,V. (1990) BioEssays 12, 409-414 20 Chen, D.J., Chan, C.S. and Pirrotta, V. (1992) Mol. Cell. Biol. 12, 598--608 21 Felsenfeld, G. (1992) Nature 355, 219-224
AT A D I S C O U N T "
T~Nos IN GENeriCS is available at a discount to all students enrolled at recognized institutions. A student subscription comprises 12 monthly issues starting from any month in 1992, Prices include air delivery worldwide. You can subscribe today by completing the form below and mailing it with your cheque (made out to Elsevier) for UK £49.00, USA and Canada $77.00, Rest of World £53.00, together with valid proof of your current student status (photocopy of student card, letter from Head of Department, etc.), to one of the addresses below. Please start my subscription to TRENOS*N GENEriCS from the next available issue (1992). I enclose my personal cheque for £ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NAME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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T/GSubscriptions, Elsevier SciencePublishers Ltd, Crown House, Linton Road, Barking, Essex, UK IGll 8JU.
"rig MAY1992 VOL. 8 NO. 5
Insistent and intransigent:a phage Mu enhancer functions in trans MARTIN R. BOOCOCK, SALLY-J. ROWLAND, W. MARSHALLSTARK AND DAVID J. SHERRAIT INSTITUTEOF GENETICS,UNIVERSITYOF GLASGOW, GLASGOW,UK Gll 5JS. In the intricate regulatory games that control gene expression, two major classes of players can be distinguished. On the one hand are cis-acting elements in the DNA and RNA sequences, such as promoters, enhancers and splice sites. To be worthy of the name, cis-acting elements are expected to interact exclusively with other genetic elements in the same DNA or RNA chain. The c/~-acting sequences are recognized and acted upon by a menagerie of freely diffusible transacting factors, including sequencespecific binding proteins, intermediary factors, and the protein and nucleoprotein machinery of transcription and RNA processing 1. In cis and in trans Why do ci~acting elements not cause regulatory chaos by interacting with DNA sequences in trans? Are they functionally incapable of unruly behaviour, or are they held in check by restrictions on access between different DNA segments? It has been suggested that a continuous DNA path between two cis-acting elements is essential for
their proper communication. However, in a number of systems where this has been tested directly, it turns out that a continuous DNA linkage is dispensable, although some elements do need to be tethered in close proximity to interact successfully 2~8. These results dealt a heavy blow to the idea that enhancers function remotely from their targets, acting either as binding sites for regulatory topoisomerases, or a& entry sites for factors that slide or assemble continuously along the DNA towards the target2, 8. In recently published work, Surette and Chaconas9 have gone a step further. They show that an enhancer element required for transposition in bacteriophage Mu can function efficiently in trans, and that it does not have to be tethered to its two target sites. The enhancer intervenes transiently in the assembly of a nucleoprotein complex between the two ends of the Mu genome, and is not involved in any of the subsequent catalytic steps of the transposition reaction (Fig. 1).
Synapsis and looping When bacteriophage Mu enters its lyric cycle, the 37 kbp proviral genome undergoes multiple rounds of replicative transposition within the chromosomal DNA of the bacterial host. Three c/s-acting elements participate in the transposition reaction: the left and right ends of the Mu genome, and an enhancer element normally located 950 bp from the left end of Mu. In addition, four proteins - MuA, MuB, IHF and HU - are required for transpositional recombination in vitro a°,l~. In the earliest detectable transposition intermediate, the type 0 complex, the MuA protein synapses the ends of the Mu genome, dividing the circular DNA into two supercoiled loops (Fig. 1). The DNA strands are then nicked to expose free 3' hydroxyl groups at the extreme left and right ends of the Mu sequences, giving a stable type I complex with one relaxed DNA loop and one supercoiled loop. The subsequent steps, in which the Mu sequences are 'pasted' into the target DNA, are interesting 12, but do not concern us here.
0 O L
R
strand ~OH R
Ip
cleavage L
R
•
Mg2+
strand transfer reaction
J
t a r g e t DNA
ancer
MuB E
substrate + MuA
hypothetical 3-site synapse
type 0 complex
E
type I complex
FIGg A simplified view of the early steps in phage Mu transposition. Arrows (L, R and E) represent the left and right Mu ends and the enhancer; shaded areas represent protein. MuA binds to multiple sites in the Mu ends and two sites in the enhancer; IHF binds to one site in the enhancer. The linking DNA sequences are not shown to scale. l~G ~AAV1992 VOL. 8 NO. 5 ©1¢~)2 Else',ier Science Publisher~, Lid (I!K)
[~]'OMMENT
•
B : ' i n a c t i "v e
C : i n a c t i v" e
FIGM
Active and inactive configurations of the Mu transposition enhancer nJ3J4. (A) Enhancer in standard configuration; (B) enhancer inverted (very low activity); (C) enhancer outside the Mu ends (inactive). For description of arrows and shaded areas, see Fig. 1. The formation of stable transposition intermediates normally requires an enhancer in cis 11,13'14, although the enhancer has not been found associated with the ends of the Mu g e n o m e in any intermediates yet characterized. The enhancer consists of an IHFbinding site w e d g e d between two binding sites for MuA. IHF is required for the enhancer to function at physiological supercoil densities, and is believed to promote interactions between the adjacent MuA sites by bending the DNA 11. Different domains of the MuA protein recognize the different sequence motifs found in the enhancer and in the ends of the Mu genome, raising the interesting possibility that individual molecules of MuA could synapse sites in the enhancer and in the Mu ends 13A4. Surette and Chaconas have now shown that the enhancer functions efficiently w h e n supplied in trans, on a 199 bp DNA fragment9. The enhancer is required only for assembly of the type 0 complex, since it can be removed by electrophoresis before formation of the type I complex.
twined configuration (Fig. 1). This specific configuration is suggested to be the basis for site selectivity in the context of a supercoiled DNA substrate. In agreement with this model, the enhancer fails to work in cis w h e n its orientation is inverted (Fig. 2B), or when it is located 'outside' the two Mu ends (Fig. 2C) 11,13,14, Surette and Chaconas argue that the local configuration postulated for the three-site synapse should be more readily accessible if the enhancer is supplied in trans (Fig. 3A). The impressive activity of the unlinked
A: Mu transposition
B: Mu Gin recombination
Selection rules The enhancer is implicated in selecting left and right Mu ends in cis in the correct head-to-head orientation3, TM. The enhancer is thought to participate in a sl'rortlived three-site synapse that precedes the type 0 complex, with the three sites adopting a specific inter-
enhancer in their experiments provides further support for the proposed intertwined three-site synapse. In trans, the enhancer works best on a short linear DNA fragment, and is less proficient when embedded in a larger linear or circular DNA; this may be due to conformational difficulties in inserting the enhancer into the synapse9. An enhancer has been implicated in the assembly of a catalytically active nucleoprotein complex in two other systems: FISdependent DNA inversion by Mu Gin, and NtrC regulation of glnA transcription6,15. These systems differ in that the enhancer functions in trans only when tethered to the target by catenation of two DNA circles (Fig. 3). For the Mu sis enhancer, simple catenation is not sufficient; the circles must be multiply entwined and supercoiled (Fig. 3B) 16, indicating that DNA topology is all-important in assembly of the three-site synapse 17. Do enhancers work well in cis simply because they are tethered close to the target site? The Mu transposition enhancer functions optimally in trans at a concentration of approximately 100 nM, but is still not as effective as an enhancer in cis 9. For comparison, DNA segments separated by 1 kbp or 10 kbp in a large nonsupercoiled circle interact at effective
C: NtrC
FIGIFI
Different degrees of tethering are required for different enhancers to function in trans. (A) Mu transposition enhancer (E) on unlinked linear DNA interacts with Mu ends (L, R) on a supercoiled substrate9 (as in Fig. 1). (B) In a supereoiled, multiply interlinked catenane, the sis enhancer stimulates recombination between the two gix sitesl6; sis and gix contain binding sites for FIS and the Gin protein of Mu, respectively. (C) In a simple catenane, the E. coli glnA enhancer stimulates the formation of an open transcription complex at the glnA promoter. NtrC protein binds at multiple sites in the enhancer; RNA polymerase with the ~54 subunit binds to the promoter to give a closed complex (,. "HG MAY1992 VOL. 8 NO. 5
I'OMMENT concentrations of approximately 60 nM and 3 nM, respectively (effective concentrations may be higher in a supercoiled molecule) TM. Hence, it is not entirely surprising that a high concentration of enhancer is required in trans., it may indicate that the interaction with the target is not controlled by single-hit kinetics, but is weak and reversible. If an enhancer DNA-protein complex can activate its target in trans, w h y is the activator protein alone not sufficient? In Mu transposition, it is evident that the MuA protomers must be delivered to the target sites in a very precise configuration that depends on IHFinduced bending of the enhancer sequences9,n. Transcriptional activator proteins from eukaryotes are generally inactive or inhibitory in isolation from their specific binding sites in DNAI; we do not yet know how they are presented to their target proteins. Do gene regulatory sequences necessarily interact in cis in the cell nucleus? In Drosophila, interactions between regulatory sequences on homologous chromosomes have been well documented for a num-
~STUDENTS
-
ber of genes, although these 'transvection' effects are not commonplace 19. Transvection at several loci requires the zeste gene product, which is believed to synapse distant chromosomal sites, bringing other regulatory sequences into closer proximity 2°. For most genes, however, a defective enhancer cannot be complemented by a functional enhancer supplied in trans on a homologOus chromosome, or 500 kbp away on the same chromosome. To understand why, we need to learn more about chromatin dynamics in the vicinity of promoters and enhancers2E
References 1 Ptashne, M. (1988) Nature 335, 683--689 2 Mt~ller, H-P~, Sogo, J.M. and Schaffner, W. (1989) Cell 58, 767-777 3 Craigie, R, and Mizuuchi, K. (1986) Cell 45, 793-800 4 Stark, W.M., Sherratt, D.J. and Boocock, M.R. (1989) Cell 58, 779--790 5 Dunaway, M. and Droge, P. (1989) Nature 341,657-659 6 Wedel, A. et al. (1990) Science 248, 486--489
SUBSCRIBE
TO
TIG
7 Rothberg, I., Hotaling, E. and Sofer, W. (1991) Nucleic Acids-Res. 19, 5713--5717 8 Plon, S.E. and Wang, J.C. (1986) Cell 45, 575-580 9 Surette, M.G. and Chaconas, G. (1992) Cell68, 1101-1108 10 Mizuuchi, K. (1983) Cell35, 785-794 11 Surette, M.G., Lavoie, B.D. and Chaconas, G. (1989) EMBOJ. 8, 3483--3489 12 Mizuuchi, K. and Adzuma, K. (1991) Cell 66, 129-140 13 Leung, P.C., Teplow, D.B. and Harshey, R.M. (1989) Nature 338, 656458 14 Mizuuchi, M. and Mizuuchi, K. (1989) Cell 58, 399~i08 15 Kanaar, R. et al. (1990) Cell 62, 353-366 16 Kanaar, R., van de Putte, P. and Cozzarelli, N.R. (1989) Cell 58, 147-149 17 Heichman, K.A. and Johnson, R.C. (1990) Science 249, 511-517 18 Wang, J.C. and Giaver, G.N. (1988) Science 240, 300-304 I 9 Pirrotta,V. (1990) BioEssays 12, 409-414 20 Chen, D.J., Chan, C.S. and Pirrotta, V. (1992) Mol. Cell. Biol. 12, 598--608 21 Felsenfeld, G. (1992) Nature 355, 219-224
AT A D I S C O U N T "
T~Nos IN GENeriCS is available at a discount to all students enrolled at recognized institutions. A student subscription comprises 12 monthly issues starting from any month in 1992, Prices include air delivery worldwide. You can subscribe today by completing the form below and mailing it with your cheque (made out to Elsevier) for UK £49.00, USA and Canada $77.00, Rest of World £53.00, together with valid proof of your current student status (photocopy of student card, letter from Head of Department, etc.), to one of the addresses below. Please start my subscription to TRENOS*N GENEriCS from the next available issue (1992). I enclose my personal cheque for £ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NAME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Remember to send valid proof of your student status with this form. Note: Current subscribers who are eligible for student discount may claim the discount only on expiry of their current subscription. No refunds, credits or extensions of the subscription period can be given. T/GSubscriptions, Journal InformationCenter, Elsevier SciencePublishing Co., Inc., 655 Avenueof the Americas, New York, NY 10010,USA.
T/GSubscriptions, Elsevier SciencePublishers Ltd, Crown House, Linton Road, Barking, Essex, UK IGll 8JU.
"rig MAY1992 VOL. 8 NO. 5
