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Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity
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control led by cell-type-specific promot ers or enhanc ers that interpret unique combinations of tr.lm.cription factors in different cell types. Studies of gene regulation in cultured cells have led to the identification of numerou~ tissue-spedfic promot ers and enhanc ers. However, it i~ becomi ng Increasingly clear that the regulatory DNA sequen ces that are Important for tmnscription in tissue culture are often different from those that direct correct ti~sue ~pecificity and tempor ospatia l re!,'lliation in l'i11(). Recent studies of gene regulation in the skeletal, cardiac and ~mooth muscle cell lineages of transgenic mice have revealed that the complete developmental expression patterns of individual muscle genes arc frequently depend ent on compos ites of indepen dent cis-acting regulatory region!'>, or mexlules, each of which directs a portion of the expression pattern of a gene. 111US, a regulatory module that directs trJn~cription in it ~uhset of mu!'>cle cells at a specific time and place in the embryo might be completely silent in other muscle cells of the same type. The strict temporospatial specificity of myogenic regulatory module s reveals surprising molecular heterog eneity among muscle cells within the ~ame lineage, and suggests the existence of myogenic subprogmms of gene expresS)(ln that arc established through comhinatorial interacti()n~ betwee n muscle-~pecific, positionally restricte d and widely express ed tmnscription factor~. Here, we review the results of several recent analyses of muscle transge ne express ion and conside r the implications of these studies for underst anding the molecular mechafllsms that generat e muscle cell diversity. Thb type of modularity of Cis-acting regulatory ~cqllences provides a means of generat ing a multitude of patterns of gene express ion from a finite numbe r of regulatory elements and is emerging as a commo n theme in the develop mental control of gene expre~sion in other cell types in organisms ranging from Drosophila to humans l .
Diversity of muscle cell types Skeletal, cardiac and smooth muscle cells originate during embryogenesIs from different mesodermal precursor cell populations. All skeletal muscle in vertebrates is TIG (.'~lynghl ~ lW~
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Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity AN11IONY B. FlRUUJ ([email protected]) ERIC N. OlSON ([email protected]) Skeleta~ cardiac and smooth muscle ceUs express overlapping sets 0/ muscle-specific genes, such that some muscle genes are expressed in only a single muscle ceQ type, whereas others are expressed in mulliple muscle ceQ Uneages. Recent studies in transgenic mice bave ret'ealed that, in many cases, mulliple, independent ds-regulatory regions, or modules, are required to direct 'he complete developmental pattern 0/ expression o/indlvidual muscle-specific genes, even within a single muscle ceQ type. The temporospattal speciflcity o/these myogenic regulatory modules is established by unique combinations o/transcription/actors and bas ret'ealed unanticipated diversity In tbe regulatory programs tbat control muscle gene expression. This type of composite regUlation 0/ muscle gene expression appears to reflect a general strategy for 'he control 0/ ceU-specific gene expression.
deriv<..>d from the S()mites, except for S()me mllscle~ in the head, which appear to arise from cephali c mes(xlerm (reviewed in Ref. 2). The somites are tran!'>ient ~tru(.1ures that form in a rostrocaudal progres~10n by segmen tation of the paraxial mesode rm adjacent to till' neural tuhe. Newly fonned somlte~ appear as paired epithelial spheres that become compar tmental ized Into a sheet of dor!'>al epithelial cells, known as the dennam yotome , which produc es muscle precurS()rs that migrdte to the limbs and body wall. Immediately heneath the de~lrnyotome is the
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myotome, which produces muscles of the back. The ventral region of the somite acquires a mesenchymal fate and fornls the sclerotome, whicll is the precursor for the ribs and vertebrae. Fast and slow muscle fibers are believed to be derived from distinct myogenic precursors3. Cardiac muscle is derived from cells in the anterior lateral plate mesoderm that converge at the ventral midline of the embryo to form a primitive heart tube, which subsequently undergoes rightward looping and eventually, formation of the atrial and ventricular chambers4. The linear heart tube is specified along its anteroposterior axis into segments that are fated to produce specific compartments of tlle mature heart, such as the outflow tract, right ventricle, left ventricle and atria 5. TIlese specific cardiac lineages are established during the initial stages of cardiogenesis by regulatory factors that remain unknown. After tile completion of cardiac looping, cardiomyocytes within the atrial and ventricular chambers express distinct sets of muscle genes that confer their unique contractile and electrophysiological properties. In contrast witll skeletal and cardiac muscle, which is derived from specific populations of mesodernlal precursors, smootll muscle arises throughout the embryo from diverse types of precursor cells, including neural crest ceJls and local mesenchymal cells derived from tile lateral mesoderm6. TIle heterogeneity observed within the smooth muscle cells (SMC<;) of the vascular, digestive, respiratory and genitourinary systems is probably a reflection of this diversity in embryonic origin. l1lere is also recent evidence that SMC') can transdifferentiate into skeletal muscle during the development of the esopha bllls7 . l1le esophagus is formed initially as smooth muscle during mouse embryogenesis. However, during late fetal development, tlle SMC') within the esophagus undergo a transition to skeletal muscle. It is unclear how this transition is initiated, whether it occurs in response to external signals or is cell autonomous, and whetller the mechanisms that regulate muscle gene expression in esophageal SMC., and skeletal muscle (:ells are the same as those in other muscle cells from these lineages.
the majority of skeletal, cardiac and smooth muscle genes, and are expressed in overlapping patterns in developing myogenic lineages] OJ]. Loss-of-function mutations in the single Me/2 gene in Drosopbila result in the absence of differentiated skeletal, cardiac and visceral muscle cells in tile embryo] 2-]4. Like other MADS-box transcription factors, members of tile MEF2 family appear to act in combination with other Iineage-restrid·ed transcription factors to control programs of gene expression. In the skeletal muscle lineage, MEF2 factors interact with members of the MyoD family of basic helix-loop-helix OJHLH) transcription factors to establish a combinatorial code that leads to activation of skeletal muscle-specific genes 15,16. TIle identities of potential MEF2 cofactors in other muscle cell lineages remain to be deternlined. Within each muscle cell lineage, individual musclespedfic genes exhibit unique temporospatial patterns of expression. TIluS, while these genes are targets for activation by myogenic transcription factors, tJley must also respond to other developmental cues, and integrate positional information in the organism witll the regulatory programs for muscle transcription. The specific transcriptional expression pattern of the gene is ultimately dependent on combinatorial interactions among the transcription factors that hind different muscle regulatory modules. TIle potential complexity of these combinatorial interactions is exemplified by the muscle creaiine kinase (Ckmm) 5' enhancer, which contains binding sites for at least eight different transcription factors 2•J7- 21 (Fig. 1). It is easy to imagine how differences in the levels of expression, or activity of individual factors, or the presence of negative regulatory factors could influence the activity of such a regulatory module, thereby conferring a unique pattern of transcription to tllat muscle gene.
Subprograms of gene regulation in skeletal muscle
Combinatorial control of muscle gen.e expression Skeletal, cardiac and smooth muscle cells express many of tJle same muscle-specific genes during embryogenesis. However, each muscle cell type also expresses unique set') of muscle-specific genes whose producte; contribute 10 their distinct contractile, metabolic and electrophysiological properties. TIle expression of a muscle gene in multiple muscle cell Iypes could, in principle, be controlled by a single set of regulatory elements that responds to a myogenic program shared by Ihe different mllscle cell Iypes or by separate regulatory modules conI rolled by regulalory factors unique to each lineage. Indeed, Ihere is evidence for both types of mecllanisms. Numerous muscle-restricted and ubiquitous transcription fa<..1ors have been shown to be important for muscle . gene activalion. However, only the myocyte enhancer fa<..tor 2 (MEF2) family of MADS (MCM1, agamous, defidens, senlm response fador)-box transcription factors has been shown to be essential for musde gene expression in all muscle celllypesH•9 . Members of the MEF2 family bind an AT-rich DNA sequence in the control regions of
Studies of the myogenic bHLH proteins, MyoD, Myf5, myogenin and Myf6, have led to a relatively simple model in which skeletal muscle genes are activated dire(:t1y by binding of tllese factors to E boxes (CANNTG) in their control regions, or indirectly by myogenic transcription factors, such as MEF2, that are themselves regulated by myogenic bHUI proteins. However, Ihis Iype of model does not readily explain the results of several recent studies that have shown that many skeletal muscle genes are controlled by mUltiple, independent, myogenic regulatory modules each of which accounts for only a portion of the full expression pattern of the gene. It also does not explain how the program for skeletal muscle transcription becomes integrated witll positional information such that a muscle enhancer might he a(.tive in only a small subset of muscle fibers at a specific time and place in the embryo. Several recent studies in Drosophila and transgenic mice exemplify tllis complexity in skeletal muscle transcription. l1le heterogeneity in muscle gene expression in Drosophila has recently been reviewed 22 , so we focus on studies in transgenic mice. TIle MJ!f5 and M.y.f6 genes are linked in a head-totail Orientation, separated by about 8 kb, in the mouse genome. During embryogenesis, Myf5 is expressed first, witll transcripts appearing in the rostral deml0myotomes at emblyonic day 8 (ER) and expression continuing in differentiating skeletal muscle· fibers. lt~yf6 is expressed transiently in the myotome between E9.0 and 11.5, and
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The existence of distinct transcriptio nal regulatory programs for I somite and limb limh myogenesis has 5' Enhancer also been suggested from studies of the mouse myogen in gene, which is controlled by a proximal skeletal muscle-specific promot er that contains an E-box and a MEF2 site.32.33 site32.33., The simplicity of myogen in regulation might reflect the fact that it is expressed in all skeletal muscles, - 1250 ----1~~~lY,i__~fr____1W___f:I!It_---1250 ---I~~r- -1050 - 1050 where it acts as a molecular switch to activate myoblast differentiation. Mutation of the MEF2 site in the myogen in promot er eliminates exMEF2 MEF2 pression of a linked lacZ /acZ reporte r gene in myogen myogeniC ic cells within the early limb limh bud hud and in a subset of cells AP2 AP2 in the somite myotome, wherea s FIGUllE 1. Regulators of the Ckmm enhancer. Expression FIGURE E-oox results in a of the Ckmm gene in skeletal mutation of the E-box muscle is controlled hy by a muscle-specific enhancer located about 1I kb upstream of delay in m.VOf.w m.'vogenin nin expression in the the gene. The diagram of this enhancer shows the locations of the known hinding binding sites for limb buds, but no effect on exthe numerous cell-type-specific and ubiquitous transcriplion 33 (Fig. 2). tion factors that, that, via combina comhinatorial pression in the somites32..33 interactions, ns. determine enhancer activity. The selective loss in activity of these mutant promoters in some skeletal muscle cells, but not in others, indilater becomes upregulated in differentiated muscle fibers cates that there is transcriptional diversity among muscle so that eventually it becomes the predominant myogenic precursor cells and suggest s that these different muscle cell bHlli gene expressed in adult skeletal muscle. muscle. The 55 5.5 kb types express different sets of transcriptional regulators. of sequen ce immediately upstream of Myj5 Myf5 is sufficient AnalysiS Analysis of regulatory elements associated with the to direct the expression of a /acZ IacZ transgene in developing myosin light chain MLCll3 MLCl/3 gene (MGD designation: craniofacial muscles, but is silent in the somites and is Mylf) in transgenic mice has also revealed a novel subsuhnot expressed in muscle muscless of the trunk until E12, when it program for muscle gene activation that could not have becomes expressed in only a subset of muscle fibers 23 . been anticipated from the expression pattern of the This region of Myf5 flanking sequence therefore contains endoge nous gene. MLCll3 transcription is control led MLC1/3 only a portion of the regulatory elements require reqUired to by two promoters, 1 F and 3r; lFand 3r~ and a muscle-specific enrecapitulate Myf5 transcription in the embryo. Similarly, hancer located about 25 kb kh downst ream of the gene· gene·H . the 6.5 kb of DNA preceding the Myj6 Myf6 gene can direct Transg Transgeenic nic mice harboring a chloramphenicol acetylexpression in skeletal muscle fibers beginn beginniing ng at E16.5, transferase (CAD reporter under control of this 3' enhanbut is inactive in the somites24 . There is also recent evi- cer and the MLG1F MLClFpromo promotter er show an intriguing pattern dence that a subset of Myf5 regulatory elements are of transgene express ion in which express ion increases contained within the Myf6 gene (P. Righy Rigby and B. Wold, Wold, in the somite myotomes along the rostrocaudal axis35 .3 6. .36. pers. commun.), which might explain why deletion mu- Because the endoge nous gene is not express ed in this tations in Myf6 lead to a loss in Myf5 My/5 express ion and type of gradient, it appear appearss that this transgene respon ds result in partial phenoc opies of Myf5 mutations in to an otherw ise cryptic regulatory program that is only mice 24-26. In the case of these two genes, which appear expose d with the combination of MLG1/3 MLCll3 regulat ory to be regulat regulateed d by multiple, indepen indepe ndent dent regulatory elements contain ed in this transgene. This evidenc e for modules, there must also be mechanisms for specific transcri ptional differences among muscles along the enhanc er-prom oter communication, becaus e a Myf5 rostrocaudal axis is consistent with earlier studies, which regulatory module within the body of the MyjO Myf6 gene showed that anterior and posterior intercostal muscles would lie closer to the promot er for Myf6 than Myf5. were distinguishable by their interactions with motor The mouse Myodl gene has also been shown to be neuron 37 s . The ability of the MLC1/3 MLCJ/3 control region to under the control of two indepe ndent muscle-specific discrim inate betwee n different muscle subtype s appear s enhancers, one at -6 kb and the other at -23 kb relative to be mediat ed by comple x interactions among numerto the transcription initiation sit 27- 30 . The -23 kb ensite e 27-30. ous transcription factors whose positive and negative hancer appears to recapitulate fully the expression patactivities are integrated by juxtaposed binding sites37 . tern of Myodl MyodJ through out embryogenesis30 , whereas The discrimination of skeletal skeletal-muscl -musclee regulatory elthe -6 kb enhanc er is express ed correctly in the somite ements betwee n different subtype s of skeletal muscle myotome, but is delayed in its activation in limb buds cells is also exemplified by the fiber-type specificity of and somites relative to the endoge nous gene ge ne 27 27 . The several muscle genes. The MLG1/3 MLCl/3 promot er and 3' -6 kb MyodJ Myodl enhanc er is also expressed preferentially enhancer, for example, direct transgene express ion to in adult fast-twitch muscle fibers 31 . Why MyodJ is con- type II fast skeletal muscle fibe~.3 ftbers38· 39 9,, while the troponin IJ trolled by two enhanc ers with overlapping expression promot er and intragenic regulatory elemen ts direct patterns is unclear. expression specifically to slow muscle fibers 40 4o . Both of
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these muscle genes have been shown to be direct target'> for activation by myogenic bHLH prot~i~. The selective accumulation of MyoD and myogenm m fast and slow adult skeletal muscle fibers, respectively, could contribute to this fiber-type specificity of gene expression31. Indeed, mutations in the E-boxes in the Ckmm enhancer disrupt expression in some skeletal muscles, but not in others41 , suggesting a differential requirement for myogenic bHLH proteins in different skeletal muscle cell types. However, that fiber-type specificity of muscle gene expression can be regulated independently of myogenic bHLH proteins has also been demonstrated for the aldolase A promoter, which is regulated in fast-twitch muscle fibers by nuclear factor 1 and a ubiquitous transcription factor MEF3, and is independent of myogenic bHLH proteins and MEF2 (Ref. 42). TIlus, there are likely to be multiple transcription factors that act through combinatorial mechanisms to control fiber-type specific gene expression.
Subprograms of gene regulation in cardiac m.uscle
Multiple subprograms for cardiac gene regulation have also been revealed by analysis of several cardiac muscle regulatory regiOns linked to lacZ transgenes. For example, desmin is expressed in developing skeletal, cardiac and smootll muscle ceUs. Within the healt, desmin is expressed homogeneously throughout the atrial and ventricular chambers. TIle 1 kb of DNA immediately preceding the desmin. gene directs the normal pattern of expression in the developing skeletal muscle lineage of transgenic mice, hut within the heart, this regulatory region is transcriptionally active only in the developing ventricles, and not in the atria4~.44. Mutation of a single MEF2 site in the desmin promoter abolished expression of the transgene in skeletal muscle and the heart43 . By contrast, mutation of an E-hox in the promoter selectively eliminated expression in skeletal muscle, but not in the heart4~. This suggests that myogenic bHLH proteins cooperate with MEF2 to activate desmin transcription in the skeletal muscle lineage, whereas in the cardiac lineage MEF2 cooperates with facto.rs that bind other regulatory elements. 'nlcse findings also suggest the existence of a specific transcriptional program for gene expression in the right ventricle. MLC113 regulatory sequences are also active in a spatially restrided manner in the developing heaJ1 that differs from that of the endogenous gene 39. A lacZ transgene under control of the MLC3F promoter and distal 3' enhancer is expressed in the presumptive left ventricle and atria, but not in the presumptive right ventricle, outflow tract, or sinus venosus where the endoge nous gene is expressed. Similarly, regulatory element'S associated with the alpha-cardiac actin gene have been shown to direct trtlllsgene expression preferentially in the left ventricle of the developing heaJ145, which contrasts with the endogenous gene, which is expressed throughout the myocardium. SM22a is a calponin-related protein expressed in developing skeletal, cardiac and smooth muscle lineage., during embryogenesis, before becoming restricted specifically to SMCs during fctal development. Whereas the endogenous SM22a gene is expressed homogeneously tJ1f<>ughout the looping heaJ1 tube, an SM22a.-I.acZ transgene containing 2.4 kh of the promoter is expressed
specifically in the developing right ventricle, but not in the developing left ventricle or atrium 46Ai . TIle regulatory factors that SUppOlt these novel programs of gene expression witJlin the developing myocardium are unknown. However, recent studies have shown that the bHLH transcription factors dJ-IAND and eHAND are expressed in specific segments of the developing heart tube that are fated to form distinct compartment,> of the heart, maldng them candidate regulators of these types of cryptic cardiogenic subprograms of gene expression 4l:lA9. Consistent with this notion, dHAND-null embryos lack a future right ventricle and show spatial distortions in cardiac gene expression within the developing cardiac tube49 .
Subprograms of gene regulation in smooth muscle
Relatively little is known of the mechanisms tJlat regulate smooth muscle gene expre')sion. However, recent studies of the SM22a gene have demonstrated that separate regulatory modules control gene expression in different SMC types. The 250 bp SM22a promoter is sufficient to recapitulate the expression pattern of the endogenous gene in arterial smooth, cardiac and skeletal muscle cells46 ,4i (Fig. 3). However, this region of the promoter is completely silent in venous SMCs and in visceral SMO, within the digestive, genitourinary, and respiratory tracts. Moreover, this regulatory region can discriminate between different types of arterial SMCs, because it is highly adive in the SMCs, within the dorsal aorta, pulmonary arteries and carotid arteries, but is inactive in the coronary arteries4 6. Presumably, SM22a: must also be controlled by SMC enhancers specific to venous, visceral and coronary artery SMC., that remain to be identified. Activation of the proximal SM22a promo ter in aJ1erial smooth, skeletal and cardiac muscle is depend ent on binding of serum response factor (SRF) to a single CARG b006 ,50. Because SRF is not muscle-specific, it must act through combinatorial mecllanisms to confer muscle specificity to the SM22a gene. Combinatorial control by the MADS-box proteins SRF and MEF2 therefore appears to be an important mecllanism for tJle regulation of muscl.e gene expression in different myogenic cell lineages.
Combinatorial codes for muscle gene activation.
Several common themes emerge from the above studies of muscle gene regulation. First, it is clear that individual muscle genes respond to multiple cues in the embryo and that their expression relies on a multiplicity of indepe ndent regulatory modules that are frequently distributed over great distances upstream, downstream and within the genes. Second, activation of muscle transcription occurs through combinatorial interactions of transcription factors. While there are certain factors that are critical for muscle gene activation, such as the myogenic bHLH factors in skeletal muscle, and MEF2 factors in multiple muscle cell types, these factors do not act alone. Rather, it is the presence or absence of other positive and negative factors that didates whether a muscle gene will be activated. 11ms, while a muscle regulatory module might contain binding sites for bHLH proteins and MEF2, the module might be transcriptionally silent in muscle cells TIG SEPTEMBER 1997 VOL. 13 No.9
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that express these factors if other positionally restricted factors that also bind the module are absent from those cells, or if inhibitory factors are present. Third, the skeletal, cardiac and smooth muscle cell lineages are highly complex and comprise distinct sublineages that can be identitind fied by novel patterns of transgene expression. The establishment of these different sublineages requires LL LL the integration of regulatory proa: MEF2 ~E MEF2 grams for muscle gene expression TATA TAT.\A TATA • TTA(J TjTA )( infonnation in the with positional information )~ I ) ( . _ . ~)(~-,--I__)~(LJ embryo. At the level of a musclespecific regulatory module, this type FIGURE 2. Combinatorial control of the myogen myogenin in promoter during mouse of regulation could be achieved myogenitr-lacZ by cooperativity between muscleemhryogenesis. (a-d) Show the expression of wild-type and mutant myogenirr-lacZ promoter transgenes in ll.s-day-old I1.S-day-old mouse emhryos. (a) Shows the wild-type pattern of pOSitionally restricted myogenirr-lacZ myogenitr-lacZ expression within each developing somite and the limb buds (LB). restricted and positionally transcription factors. In most cases (b) Myogenic hHLH regulatory factors (MRFs) bind as heterodimers with E-proteins to the in which novel myogenic sublinE-box in the proximal promoter. Mutation of the E-box causes a delay in the upregulation eages have been revealed by patmyogenitr-lacZ expression expres~ion within the limb buds but does not effect somite expression. of myogenirr-lacZ (c) Mutation of the MEF2 site eliminates myogenirr-lacZlimb myogenitr-lacZlimb bud expression and terns of trans transgene gene expression, the subset of cells within the myotome. (d) Mutation of the E-box and exad expression within a sub..'iet exact combination of cis-elements MEF2 site eliminates all a\l expression of myogenifl-lacZ. myogenirr/acZ. and transcription factors required for that pattern is unknown. Hox gene products are attractive candidates for positional regulation of muscle gene expression, and there is evidence for a role of such fadors factors in muscle gene regulation'il, but, to date, Hox gene products have not been shown to interact directly with myogenic regulatory modules to confer positional specificity. A relevant question in the above types of studies is whether a novel pattern of gene expression observed with a transgene indeed reflects a portion of the normal regulatory inputs of the endogenous gene or whether the combination of regulatelemento; within the transgene tJ"'ansgene ory elements has generated a novel pattern of gene expression unrelated to that of the normal nonnal gene. While such studies have generally been interpreted to indicate the existence of independent regulatory modules, the sum of which supports the full expression associated gene, it is pattern of the aSSOciated also pOSSible possible that chromatin conformation and precise spatial arrangements between promoters and enhancers are important for the correct FIGURE 3. Expression of the SM22-1acZ SM22-lacZ transgene. The 1343 bp promoter of the temporospatial expression patterns SM22a gene was linked to lacz lacZ and used to create transgenic mice46 . (a) Whole mount of muscle-specific of genes. Thus, when a 12.5-day-old 12';-clay-old embryo showing shOWing SM22a-lacZ expression within the developing promoters and enhancers are sepavasculature, heart and somite myotomes (M). (b) Expression within the heart at this from their surrounding serated time point shows compartmentalized expression with high levels of SM22a-lacZ (A). quences and their orientations are expression in the right ventricle (RV) but not within the left ventricle (LV) or atria (A). SM22a-lacZ expression is also strong within the dorsal aorta (DA). (c, d) Transverse altered relative to those in the native sections through the esophagus (E) and dorsal aorta (DA) clearly show the specificity of gene, reproducible repnxludble alterations in their this transcriptional control module for arterial, but not visceral, smooth muscle. muscle. transcriptional specificity could result.
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Evolution of muscle cell diversity through modular enhancers
11 Ticho, B.S., Stainer, Y.R., Fishman, M.e. and Breitbart, R.E. (19%) Mecb. Dev. 59, 205-2]8 12 Lilly, B. et al. (1995) Science 267, 688-693 13 Bour, B.A. el al. (1995) Ge11es Dev. 9, 730-74] 14 Ranganayakulu, G. et al. (]995) Dev. BioI. 171. 169-181 15 Molkentin, ].D., Black, B.L., Martin, J.F. and Olson, E.N. (1995) Ce1l83, 1125-1136
Modularity of gene regulation in which multiple independent regulatory regions are required to generate the complete expression pattern of a gene during embryonic development and adulthood is not unique to muscle cells and has been carefully documented for numerous other types of genes in diverse organisms, particularly in Drosophila and sea urchin embryos (reviewed in Ref. 1). Why have muscle genes evolved SUcil complex sets of independent regulatory modules rather than having single enhancers for the control of expression in all muscle cells of a particular type? One possibility is that this type of modular regulation of gene expression provides greater flexibility to transcriptional control than might be achieved through a single regulatory module. In addition, modularity of transcriptional control sequences facilitates the establishment of complex patterns of gene transcription during evolution. Thus, in a simple organism with a relatively low complexity of muscle types, a single regulatory module might be sufficient to direct the complete expression pattern of a muscle gene, whereas the activation of muscle gene expression in more complex organisms could be achieved by the duplication of modules and concomitant recruitment of additional regulatory elements that confer positional specifiCity. The dependence of myogenic regulatory modules on combinations of cis elements makes it possible to create novel pat1erns of gene expression by combining different regulatory elements. As more myogenic regulatolY modules are identified and their regulatory factors are characterized, it should be possible to design myogenic regulatory modules that confer precise patterns of temporospatial regulation of muscle gene expression by combining regulatolY factor binding sites. 'J11is will then provide the opportunity to express exogenolls genes se)e<.1ively in different types of muscle cells ~Jt specific limes in devclopmcnt.
16 Kaushal, S., Schneider, J.W., Nadal-Ginard, B. and Mahdavi, Y. (1994) Science266, 1236-]24.0 17 Lassal', A.B. et al. (1989) Ce1/58, 823-831 Mol. Cell. Bioi. 13, 2753-2764
20 Fabre-Suver, C. and Hauschka, S.D. (996) J. BioI. Cbem. 271,4646-4652
O;erjesi, P., Ully, B., Hinkley, C., Perry, M. and Olson, E.N. (1994) J. BioI. Cbem. 269, 16740-]6745 22 Donoghue, M]. and Sanes, J.R. (] 994) Trends Ge1tel. 10,
21
396-401
(1993) DeIJelopmenl118, 61-69 24 Braun, T. et al. (1994) Development 120,3083-3092 25 Patapoutian, A. et al. (l995) Development ]21,3347-3358
26 Olson, E.N., Arnold, Ii.H., Rigby, P.W. and Wold, BJ. (]996) Cel/85, 1-4
27 Asakura, A., Lyons. G.E. and Tapscott, S.J. (]99,) Dev. BioI. 17], 386-398
Faennan, A. and Shani, M. (]993) DevelojJment 118, 919-929 29 Goldhamer, DJ. et al. (995) Development] 2], 637-649 30 Goldhamer, DJ., Faetman, A., Shani, M. and Emerson. e.P., Jr (}992) Science 256, 538-542 31 Hughes, S.M. et al. (993) DelJelojJme1t1 118, 1137-1147 32 Cheng, T.e., Wallace, M.e., Mel'lie, J.P. and Olson, E.N.
28
(993) Science 26], 215-28
33 Vee, S.P. and Rigby, P.W. (]993) Genes Det). 7, 12n-1289 34
7 Patapollti:l11, A., \'(/old. B.J. and \X':lgner. itA. (]99S) Sc;iemce270. ]H]H-JH2]
8
Donoghue, M. el al. (]988) Genes Dev. 2, ]n9-1790
35 Grieshammer, u., Sassoon, D. and Rosenthal, N. (1992) Ce1l69, 79-93 36 Donoghue. M.J. et al. (992) Ce1l69, 67-77
37 Rao, M.\l., Donoghuc, M.J., Merlie, .J.P. and Sanes, J.R. (1996) Mol. Cell. BioI. 16,3909-3922
38 McGrew. M.J. et al. (J996) Mol. Cell. BioI. ]6, 4524-4534 39 Kclly, R elal. (995)]. Cell. BioI. 129, 383-396
We thank members of our laboratory for helpful COI11ments, and 11. Tii'.enor (lnd 11. Hawlf for assi~1~mcc with gr'dphic'i. Work in our laboratory ill supported by gmntll from 'J11c NatjonaJ Institut.es of Health, 'J11e American He.lIi Associclliot1, ')11e Hohe!1 A. Wekh Foundation, the HlIll1~lJ1 Frontiers Sciences Program
Patapoutian, A., Miner, J.H., Lyons, G.E. and Wold, B.
23
Acknowledgements
References
Mueller, P.R. and Wold. B. (]989) SCience 246, 780-786 Amacher, S.L., Buskin, J.N. and Hauschka, S.D. (993)
18 19
40 Banerjec-Basu, S. and Buonanno, A. (1993) Mol. Cel/. BioI. 13, 7019-7028 41 Shield, M.A., Haugen, H.S., Clegg, e.H. and Hauschka, S.D. 09%) Mol. Cel/. BioI. ]6,5058-5068 42 Spitz, F., Salminen, M., Dcmingnon, J" Kahn, A., Daegelen, D. and Maire, P. (997) Mol. Cell. 13;01. 17. 656-666 . 43 Kuisk, J.Jt, U, H., Tran, D. and Capetanaki, Y. (]996) De". BioI. 174. ]-]3 44 Li. Z., March:md. P., Humbert, J., Babinel, C. and Pat!lin. D. (993) J)(!tJelojJm(Jl111 ]7, 947-959 45 Bihen, e., Hadchouel,,1., Tajhakhsh, S. and Bllckingham, M. (J996) Det). BioI. 173, 200-2]2 46 Li. 1.., Miano,,1.M., Mercer, B. and Olson, E.N. (996) .f. Cel/. BioI. 132, H49-859 47 Kim, S. el al. (997) Mol. Cel/. BioI. 17.2266-2278 48 SrivtlSI:1V:I, D., C'\crjcsi, P. and Olson. E.N. (995) Sciel1ce 270, ]995-]999 49 Srivastava, D., 11101113S, T., Un, Q. and Olson. E.N. Nal. G(!11el. (in press) . . 50 Li. I.. el al. (]997) Dell. BioI. ]87, 31 ]-32] 51 Olson. E.N. and Hosenthal, N. (J994) Ce1l79, 9-]2
"ossell, l..A., KelVin, 0 ..1., Sternberg. E.A. and Olson. E.N. (JI)H9) Mol. O!//. mol. 1),5022-5033
9 Olson, E.N., Perry. M. and S('hlllz. itA. (1995) Dlm. BioI. ]72,2-11 10 Eclmondson, I)'(~" Lyons. ('.E., M:111in. ,l.F. mltl Olson. E.N. (]CJJ.i) /)(JwlojJl1wnl ]20.125]-]263
A.n. Ff.,."llf flIt-dE.N. 01.~o" am i11. tbe JJepfll1mm1.f of /v1oleculm' Biology and Oncology,Hamon CelU(!1'for Ba.";c CanceI'Rese(.l1·cb, 71le 1111itX!/'si{1' ofT(J:X:os Sotllbweslem Medical Cel11(Jl'af Dallas, Dallas, TX 75235-9148, USA.
TIG SEPTEMBER 1997 VOL. 13 No.9
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26 Wolfe. K H. and Shields. D.C (}9<)7) .Yat/lre 3~r'. 70H-7U 27 Lundin. LO (1lJ'-)j) Gellom icsI6.1- 19 28 Garda-Femandl'z. J. and Holland. I' W. ( 1(94) Naill re
:\70.
40 Patel. K et at 09<)(}) [Jel'. BIoi 178. 327-.-\42 41 Nakamllf'J, T.. Sugino. K.. Titani. K. and Sligino. H. (1991> ] Bioi Chem 266. 194.32-19437 42 Zimmennan. L 13 . De Jesll~-EM'Uhar. J.M. and Harland, R.M. <19%) eel/B(,. '5l)
'56~'56()
29 Storm. E.E. el al (1994) ,Yallln! ~,(~. 639-64.3 30 LI. X. and :-'011.1\1. (1994) ,\·(lllIr(':\67. H,3--8"" 31 Dc Beer. G.
(1910) Emln)'os alld Allceslor s,
Cl.lrl'ndon Pre~~ 32 Haffter. P. el til. (19%) ["JeI>e/opmell/1.B, 1-:16 33 Joyner, A (1996) 1h'I/(ls (;ell£'t 12, 1-;""'20 34 I.\'on~, K 1\1 ,J()nl'~, C\I and Hogan. IH.M (1991) 'f'rl'lItis Gellel 7. 40H-i 12
J. Cooke
is 111 the Dit'isioll of Del'elopmelltat Nellrobiologv, Sa/lOlll ll IlIslilllle for Jfedica l Research. The RitiRell'ay.
35 Sld()\\. A. (1992) Pmc. ,\'all Acad. So. (' S A. H9, '509B-'i102 36 \X'ur~t. W. Allerharh. A B and Joyner. A.L. (1994)
Mrlllfill . LOlldol/. eK i\lF71M M.A. Nowak alld M. Boerlljst are m Ihe lJepartm ent of ZOO/OlD'. SO/llh Parks Road. Oxford. UK OX I 3P J. Maynard-Smith IS ill /he Sch(Jol oj BIOI{)J,1iclIl SCIences. r:llil'ersIZI' o/SUSS(!x. Falmer. Bnp,h/o ll. {'K B:../1 9QG
["JeI'e/opmeIll120.206-;.....2075 37 L:.unb. T M. et til (199:1) SCience 262. 71j-71R
38 Sa~;II. Y. 1'1 (/1 (199'1) .\'alllr£'3 76 . .~:\j-.:t~6 39 Hellllll,ltl-i3manloli. A . Kelly. (Hi and Melton. 0 A ( 19<)1) Cell ....... 2R'}-29S
Tran~criptional regulation of tis1>ue-spedfic genes is
control led by cell-type-specific promot ers or enhanc ers that interpret unique combinations of tr.lm.cription factors in different cell types. Studies of gene regulation in cultured cells have led to the identification of numerou~ tissue-spedfic promot ers and enhanc ers. However, it i~ becomi ng Increasingly clear that the regulatory DNA sequen ces that are Important for tmnscription in tissue culture are often different from those that direct correct ti~sue ~pecificity and tempor ospatia l re!,'lliation in l'i11(). Recent studies of gene regulation in the skeletal, cardiac and ~mooth muscle cell lineages of transgenic mice have revealed that the complete developmental expression patterns of individual muscle genes arc frequently depend ent on compos ites of indepen dent cis-acting regulatory region!'>, or mexlules, each of which directs a portion of the expression pattern of a gene. 111US, a regulatory module that directs trJn~cription in it ~uhset of mu!'>cle cells at a specific time and place in the embryo might be completely silent in other muscle cells of the same type. The strict temporospatial specificity of myogenic regulatory module s reveals surprising molecular heterog eneity among muscle cells within the ~ame lineage, and suggests the existence of myogenic subprogmms of gene expresS)(ln that arc established through comhinatorial interacti()n~ betwee n muscle-~pecific, positionally restricte d and widely express ed tmnscription factor~. Here, we review the results of several recent analyses of muscle transge ne express ion and conside r the implications of these studies for underst anding the molecular mechafllsms that generat e muscle cell diversity. Thb type of modularity of Cis-acting regulatory ~cqllences provides a means of generat ing a multitude of patterns of gene express ion from a finite numbe r of regulatory elements and is emerging as a commo n theme in the develop mental control of gene expre~sion in other cell types in organisms ranging from Drosophila to humans l .
Diversity of muscle cell types Skeletal, cardiac and smooth muscle cells originate during embryogenesIs from different mesodermal precursor cell populations. All skeletal muscle in vertebrates is TIG (.'~lynghl ~ lW~
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Modular regulation of muscle gene transcription: a mechanism for muscle cell diversity AN11IONY B. FlRUUJ ([email protected]) ERIC N. OlSON ([email protected]) Skeleta~ cardiac and smooth muscle ceUs express overlapping sets 0/ muscle-specific genes, such that some muscle genes are expressed in only a single muscle ceQ type, whereas others are expressed in mulliple muscle ceQ Uneages. Recent studies in transgenic mice bave ret'ealed that, in many cases, mulliple, independent ds-regulatory regions, or modules, are required to direct 'he complete developmental pattern 0/ expression o/indlvidual muscle-specific genes, even within a single muscle ceQ type. The temporospattal speciflcity o/these myogenic regulatory modules is established by unique combinations o/transcription/actors and bas ret'ealed unanticipated diversity In tbe regulatory programs tbat control muscle gene expression. This type of composite regUlation 0/ muscle gene expression appears to reflect a general strategy for 'he control 0/ ceU-specific gene expression.
deriv<..>d from the S()mites, except for S()me mllscle~ in the head, which appear to arise from cephali c mes(xlerm (reviewed in Ref. 2). The somites are tran!'>ient ~tru(.1ures that form in a rostrocaudal progres~10n by segmen tation of the paraxial mesode rm adjacent to till' neural tuhe. Newly fonned somlte~ appear as paired epithelial spheres that become compar tmental ized Into a sheet of dor!'>al epithelial cells, known as the dennam yotome , which produc es muscle precurS()rs that migrdte to the limbs and body wall. Immediately heneath the de~lrnyotome is the
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myotome, which produces muscles of the back. The ventral region of the somite acquires a mesenchymal fate and fornls the sclerotome, whicll is the precursor for the ribs and vertebrae. Fast and slow muscle fibers are believed to be derived from distinct myogenic precursors3. Cardiac muscle is derived from cells in the anterior lateral plate mesoderm that converge at the ventral midline of the embryo to form a primitive heart tube, which subsequently undergoes rightward looping and eventually, formation of the atrial and ventricular chambers4. The linear heart tube is specified along its anteroposterior axis into segments that are fated to produce specific compartments of tlle mature heart, such as the outflow tract, right ventricle, left ventricle and atria 5. TIlese specific cardiac lineages are established during the initial stages of cardiogenesis by regulatory factors that remain unknown. After tile completion of cardiac looping, cardiomyocytes within the atrial and ventricular chambers express distinct sets of muscle genes that confer their unique contractile and electrophysiological properties. In contrast witll skeletal and cardiac muscle, which is derived from specific populations of mesodernlal precursors, smootll muscle arises throughout the embryo from diverse types of precursor cells, including neural crest ceJls and local mesenchymal cells derived from tile lateral mesoderm6. TIle heterogeneity observed within the smooth muscle cells (SMC<;) of the vascular, digestive, respiratory and genitourinary systems is probably a reflection of this diversity in embryonic origin. l1lere is also recent evidence that SMC') can transdifferentiate into skeletal muscle during the development of the esopha bllls7 . l1le esophagus is formed initially as smooth muscle during mouse embryogenesis. However, during late fetal development, tlle SMC') within the esophagus undergo a transition to skeletal muscle. It is unclear how this transition is initiated, whether it occurs in response to external signals or is cell autonomous, and whetller the mechanisms that regulate muscle gene expression in esophageal SMC., and skeletal muscle (:ells are the same as those in other muscle cells from these lineages.
the majority of skeletal, cardiac and smooth muscle genes, and are expressed in overlapping patterns in developing myogenic lineages] OJ]. Loss-of-function mutations in the single Me/2 gene in Drosopbila result in the absence of differentiated skeletal, cardiac and visceral muscle cells in tile embryo] 2-]4. Like other MADS-box transcription factors, members of tile MEF2 family appear to act in combination with other Iineage-restrid·ed transcription factors to control programs of gene expression. In the skeletal muscle lineage, MEF2 factors interact with members of the MyoD family of basic helix-loop-helix OJHLH) transcription factors to establish a combinatorial code that leads to activation of skeletal muscle-specific genes 15,16. TIle identities of potential MEF2 cofactors in other muscle cell lineages remain to be deternlined. Within each muscle cell lineage, individual musclespedfic genes exhibit unique temporospatial patterns of expression. TIluS, while these genes are targets for activation by myogenic transcription factors, tJley must also respond to other developmental cues, and integrate positional information in the organism witll the regulatory programs for muscle transcription. The specific transcriptional expression pattern of the gene is ultimately dependent on combinatorial interactions among the transcription factors that hind different muscle regulatory modules. TIle potential complexity of these combinatorial interactions is exemplified by the muscle creaiine kinase (Ckmm) 5' enhancer, which contains binding sites for at least eight different transcription factors 2•J7- 21 (Fig. 1). It is easy to imagine how differences in the levels of expression, or activity of individual factors, or the presence of negative regulatory factors could influence the activity of such a regulatory module, thereby conferring a unique pattern of transcription to tllat muscle gene.
Subprograms of gene regulation in skeletal muscle
Combinatorial control of muscle gen.e expression Skeletal, cardiac and smooth muscle cells express many of tJle same muscle-specific genes during embryogenesis. However, each muscle cell type also expresses unique set') of muscle-specific genes whose producte; contribute 10 their distinct contractile, metabolic and electrophysiological properties. TIle expression of a muscle gene in multiple muscle cell Iypes could, in principle, be controlled by a single set of regulatory elements that responds to a myogenic program shared by Ihe different mllscle cell Iypes or by separate regulatory modules conI rolled by regulalory factors unique to each lineage. Indeed, Ihere is evidence for both types of mecllanisms. Numerous muscle-restricted and ubiquitous transcription fa<..1ors have been shown to be important for muscle . gene activalion. However, only the myocyte enhancer fa<..tor 2 (MEF2) family of MADS (MCM1, agamous, defidens, senlm response fador)-box transcription factors has been shown to be essential for musde gene expression in all muscle celllypesH•9 . Members of the MEF2 family bind an AT-rich DNA sequence in the control regions of
Studies of the myogenic bHLH proteins, MyoD, Myf5, myogenin and Myf6, have led to a relatively simple model in which skeletal muscle genes are activated dire(:t1y by binding of tllese factors to E boxes (CANNTG) in their control regions, or indirectly by myogenic transcription factors, such as MEF2, that are themselves regulated by myogenic bHUI proteins. However, Ihis Iype of model does not readily explain the results of several recent studies that have shown that many skeletal muscle genes are controlled by mUltiple, independent, myogenic regulatory modules each of which accounts for only a portion of the full expression pattern of the gene. It also does not explain how the program for skeletal muscle transcription becomes integrated witll positional information such that a muscle enhancer might he a(.tive in only a small subset of muscle fibers at a specific time and place in the embryo. Several recent studies in Drosophila and transgenic mice exemplify tllis complexity in skeletal muscle transcription. l1le heterogeneity in muscle gene expression in Drosophila has recently been reviewed 22 , so we focus on studies in transgenic mice. TIle MJ!f5 and M.y.f6 genes are linked in a head-totail Orientation, separated by about 8 kb, in the mouse genome. During embryogenesis, Myf5 is expressed first, witll transcripts appearing in the rostral deml0myotomes at emblyonic day 8 (ER) and expression continuing in differentiating skeletal muscle· fibers. lt~yf6 is expressed transiently in the myotome between E9.0 and 11.5, and
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The existence of distinct transcriptio nal regulatory programs for I somite and limb limh myogenesis has 5' Enhancer also been suggested from studies of the mouse myogen in gene, which is controlled by a proximal skeletal muscle-specific promot er that contains an E-box and a MEF2 site.32.33 site32.33., The simplicity of myogen in regulation might reflect the fact that it is expressed in all skeletal muscles, - 1250 ----1~~~lY,i__~fr____1W___f:I!It_---1250 ---I~~r- -1050 - 1050 where it acts as a molecular switch to activate myoblast differentiation. Mutation of the MEF2 site in the myogen in promot er eliminates exMEF2 MEF2 pression of a linked lacZ /acZ reporte r gene in myogen myogeniC ic cells within the early limb limh bud hud and in a subset of cells AP2 AP2 in the somite myotome, wherea s FIGUllE 1. Regulators of the Ckmm enhancer. Expression FIGURE E-oox results in a of the Ckmm gene in skeletal mutation of the E-box muscle is controlled hy by a muscle-specific enhancer located about 1I kb upstream of delay in m.VOf.w m.'vogenin nin expression in the the gene. The diagram of this enhancer shows the locations of the known hinding binding sites for limb buds, but no effect on exthe numerous cell-type-specific and ubiquitous transcriplion 33 (Fig. 2). tion factors that, that, via combina comhinatorial pression in the somites32..33 interactions, ns. determine enhancer activity. The selective loss in activity of these mutant promoters in some skeletal muscle cells, but not in others, indilater becomes upregulated in differentiated muscle fibers cates that there is transcriptional diversity among muscle so that eventually it becomes the predominant myogenic precursor cells and suggest s that these different muscle cell bHlli gene expressed in adult skeletal muscle. muscle. The 55 5.5 kb types express different sets of transcriptional regulators. of sequen ce immediately upstream of Myj5 Myf5 is sufficient AnalysiS Analysis of regulatory elements associated with the to direct the expression of a /acZ IacZ transgene in developing myosin light chain MLCll3 MLCl/3 gene (MGD designation: craniofacial muscles, but is silent in the somites and is Mylf) in transgenic mice has also revealed a novel subsuhnot expressed in muscle muscless of the trunk until E12, when it program for muscle gene activation that could not have becomes expressed in only a subset of muscle fibers 23 . been anticipated from the expression pattern of the This region of Myf5 flanking sequence therefore contains endoge nous gene. MLCll3 transcription is control led MLC1/3 only a portion of the regulatory elements require reqUired to by two promoters, 1 F and 3r; lFand 3r~ and a muscle-specific enrecapitulate Myf5 transcription in the embryo. Similarly, hancer located about 25 kb kh downst ream of the gene· gene·H . the 6.5 kb of DNA preceding the Myj6 Myf6 gene can direct Transg Transgeenic nic mice harboring a chloramphenicol acetylexpression in skeletal muscle fibers beginn beginniing ng at E16.5, transferase (CAD reporter under control of this 3' enhanbut is inactive in the somites24 . There is also recent evi- cer and the MLG1F MLClFpromo promotter er show an intriguing pattern dence that a subset of Myf5 regulatory elements are of transgene express ion in which express ion increases contained within the Myf6 gene (P. Righy Rigby and B. Wold, Wold, in the somite myotomes along the rostrocaudal axis35 .3 6. .36. pers. commun.), which might explain why deletion mu- Because the endoge nous gene is not express ed in this tations in Myf6 lead to a loss in Myf5 My/5 express ion and type of gradient, it appear appearss that this transgene respon ds result in partial phenoc opies of Myf5 mutations in to an otherw ise cryptic regulatory program that is only mice 24-26. In the case of these two genes, which appear expose d with the combination of MLG1/3 MLCll3 regulat ory to be regulat regulateed d by multiple, indepen indepe ndent dent regulatory elements contain ed in this transgene. This evidenc e for modules, there must also be mechanisms for specific transcri ptional differences among muscles along the enhanc er-prom oter communication, becaus e a Myf5 rostrocaudal axis is consistent with earlier studies, which regulatory module within the body of the MyjO Myf6 gene showed that anterior and posterior intercostal muscles would lie closer to the promot er for Myf6 than Myf5. were distinguishable by their interactions with motor The mouse Myodl gene has also been shown to be neuron 37 s . The ability of the MLC1/3 MLCJ/3 control region to under the control of two indepe ndent muscle-specific discrim inate betwee n different muscle subtype s appear s enhancers, one at -6 kb and the other at -23 kb relative to be mediat ed by comple x interactions among numerto the transcription initiation sit 27- 30 . The -23 kb ensite e 27-30. ous transcription factors whose positive and negative hancer appears to recapitulate fully the expression patactivities are integrated by juxtaposed binding sites37 . tern of Myodl MyodJ through out embryogenesis30 , whereas The discrimination of skeletal skeletal-muscl -musclee regulatory elthe -6 kb enhanc er is express ed correctly in the somite ements betwee n different subtype s of skeletal muscle myotome, but is delayed in its activation in limb buds cells is also exemplified by the fiber-type specificity of and somites relative to the endoge nous gene ge ne 27 27 . The several muscle genes. The MLG1/3 MLCl/3 promot er and 3' -6 kb MyodJ Myodl enhanc er is also expressed preferentially enhancer, for example, direct transgene express ion to in adult fast-twitch muscle fibers 31 . Why MyodJ is con- type II fast skeletal muscle fibe~.3 ftbers38· 39 9,, while the troponin IJ trolled by two enhanc ers with overlapping expression promot er and intragenic regulatory elemen ts direct patterns is unclear. expression specifically to slow muscle fibers 40 4o . Both of
oo kb kb I
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these muscle genes have been shown to be direct target'> for activation by myogenic bHLH prot~i~. The selective accumulation of MyoD and myogenm m fast and slow adult skeletal muscle fibers, respectively, could contribute to this fiber-type specificity of gene expression31. Indeed, mutations in the E-boxes in the Ckmm enhancer disrupt expression in some skeletal muscles, but not in others41 , suggesting a differential requirement for myogenic bHLH proteins in different skeletal muscle cell types. However, that fiber-type specificity of muscle gene expression can be regulated independently of myogenic bHLH proteins has also been demonstrated for the aldolase A promoter, which is regulated in fast-twitch muscle fibers by nuclear factor 1 and a ubiquitous transcription factor MEF3, and is independent of myogenic bHLH proteins and MEF2 (Ref. 42). TIlus, there are likely to be multiple transcription factors that act through combinatorial mechanisms to control fiber-type specific gene expression.
Subprograms of gene regulation in cardiac m.uscle
Multiple subprograms for cardiac gene regulation have also been revealed by analysis of several cardiac muscle regulatory regiOns linked to lacZ transgenes. For example, desmin is expressed in developing skeletal, cardiac and smootll muscle ceUs. Within the healt, desmin is expressed homogeneously throughout the atrial and ventricular chambers. TIle 1 kb of DNA immediately preceding the desmin. gene directs the normal pattern of expression in the developing skeletal muscle lineage of transgenic mice, hut within the heart, this regulatory region is transcriptionally active only in the developing ventricles, and not in the atria4~.44. Mutation of a single MEF2 site in the desmin promoter abolished expression of the transgene in skeletal muscle and the heart43 . By contrast, mutation of an E-hox in the promoter selectively eliminated expression in skeletal muscle, but not in the heart4~. This suggests that myogenic bHLH proteins cooperate with MEF2 to activate desmin transcription in the skeletal muscle lineage, whereas in the cardiac lineage MEF2 cooperates with facto.rs that bind other regulatory elements. 'nlcse findings also suggest the existence of a specific transcriptional program for gene expression in the right ventricle. MLC113 regulatory sequences are also active in a spatially restrided manner in the developing heaJ1 that differs from that of the endogenous gene 39. A lacZ transgene under control of the MLC3F promoter and distal 3' enhancer is expressed in the presumptive left ventricle and atria, but not in the presumptive right ventricle, outflow tract, or sinus venosus where the endoge nous gene is expressed. Similarly, regulatory element'S associated with the alpha-cardiac actin gene have been shown to direct trtlllsgene expression preferentially in the left ventricle of the developing heaJ145, which contrasts with the endogenous gene, which is expressed throughout the myocardium. SM22a is a calponin-related protein expressed in developing skeletal, cardiac and smooth muscle lineage., during embryogenesis, before becoming restricted specifically to SMCs during fctal development. Whereas the endogenous SM22a gene is expressed homogeneously tJ1f<>ughout the looping heaJ1 tube, an SM22a.-I.acZ transgene containing 2.4 kh of the promoter is expressed
specifically in the developing right ventricle, but not in the developing left ventricle or atrium 46Ai . TIle regulatory factors that SUppOlt these novel programs of gene expression witJlin the developing myocardium are unknown. However, recent studies have shown that the bHLH transcription factors dJ-IAND and eHAND are expressed in specific segments of the developing heart tube that are fated to form distinct compartment,> of the heart, maldng them candidate regulators of these types of cryptic cardiogenic subprograms of gene expression 4l:lA9. Consistent with this notion, dHAND-null embryos lack a future right ventricle and show spatial distortions in cardiac gene expression within the developing cardiac tube49 .
Subprograms of gene regulation in smooth muscle
Relatively little is known of the mechanisms tJlat regulate smooth muscle gene expre')sion. However, recent studies of the SM22a gene have demonstrated that separate regulatory modules control gene expression in different SMC types. The 250 bp SM22a promoter is sufficient to recapitulate the expression pattern of the endogenous gene in arterial smooth, cardiac and skeletal muscle cells46 ,4i (Fig. 3). However, this region of the promoter is completely silent in venous SMCs and in visceral SMO, within the digestive, genitourinary, and respiratory tracts. Moreover, this regulatory region can discriminate between different types of arterial SMCs, because it is highly adive in the SMCs, within the dorsal aorta, pulmonary arteries and carotid arteries, but is inactive in the coronary arteries4 6. Presumably, SM22a: must also be controlled by SMC enhancers specific to venous, visceral and coronary artery SMC., that remain to be identified. Activation of the proximal SM22a promo ter in aJ1erial smooth, skeletal and cardiac muscle is depend ent on binding of serum response factor (SRF) to a single CARG b006 ,50. Because SRF is not muscle-specific, it must act through combinatorial mecllanisms to confer muscle specificity to the SM22a gene. Combinatorial control by the MADS-box proteins SRF and MEF2 therefore appears to be an important mecllanism for tJle regulation of muscl.e gene expression in different myogenic cell lineages.
Combinatorial codes for muscle gene activation.
Several common themes emerge from the above studies of muscle gene regulation. First, it is clear that individual muscle genes respond to multiple cues in the embryo and that their expression relies on a multiplicity of indepe ndent regulatory modules that are frequently distributed over great distances upstream, downstream and within the genes. Second, activation of muscle transcription occurs through combinatorial interactions of transcription factors. While there are certain factors that are critical for muscle gene activation, such as the myogenic bHLH factors in skeletal muscle, and MEF2 factors in multiple muscle cell types, these factors do not act alone. Rather, it is the presence or absence of other positive and negative factors that didates whether a muscle gene will be activated. 11ms, while a muscle regulatory module might contain binding sites for bHLH proteins and MEF2, the module might be transcriptionally silent in muscle cells TIG SEPTEMBER 1997 VOL. 13 No.9
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that express these factors if other positionally restricted factors that also bind the module are absent from those cells, or if inhibitory factors are present. Third, the skeletal, cardiac and smooth muscle cell lineages are highly complex and comprise distinct sublineages that can be identitind fied by novel patterns of transgene expression. The establishment of these different sublineages requires LL LL the integration of regulatory proa: MEF2 ~E MEF2 grams for muscle gene expression TATA TAT.\A TATA • TTA(J TjTA )( infonnation in the with positional information )~ I ) ( . _ . ~)(~-,--I__)~(LJ embryo. At the level of a musclespecific regulatory module, this type FIGURE 2. Combinatorial control of the myogen myogenin in promoter during mouse of regulation could be achieved myogenitr-lacZ by cooperativity between muscleemhryogenesis. (a-d) Show the expression of wild-type and mutant myogenirr-lacZ promoter transgenes in ll.s-day-old I1.S-day-old mouse emhryos. (a) Shows the wild-type pattern of pOSitionally restricted myogenirr-lacZ myogenitr-lacZ expression within each developing somite and the limb buds (LB). restricted and positionally transcription factors. In most cases (b) Myogenic hHLH regulatory factors (MRFs) bind as heterodimers with E-proteins to the in which novel myogenic sublinE-box in the proximal promoter. Mutation of the E-box causes a delay in the upregulation eages have been revealed by patmyogenitr-lacZ expression expres~ion within the limb buds but does not effect somite expression. of myogenirr-lacZ (c) Mutation of the MEF2 site eliminates myogenirr-lacZlimb myogenitr-lacZlimb bud expression and terns of trans transgene gene expression, the subset of cells within the myotome. (d) Mutation of the E-box and exad expression within a sub..'iet exact combination of cis-elements MEF2 site eliminates all a\l expression of myogenifl-lacZ. myogenirr/acZ. and transcription factors required for that pattern is unknown. Hox gene products are attractive candidates for positional regulation of muscle gene expression, and there is evidence for a role of such fadors factors in muscle gene regulation'il, but, to date, Hox gene products have not been shown to interact directly with myogenic regulatory modules to confer positional specificity. A relevant question in the above types of studies is whether a novel pattern of gene expression observed with a transgene indeed reflects a portion of the normal regulatory inputs of the endogenous gene or whether the combination of regulatelemento; within the transgene tJ"'ansgene ory elements has generated a novel pattern of gene expression unrelated to that of the normal nonnal gene. While such studies have generally been interpreted to indicate the existence of independent regulatory modules, the sum of which supports the full expression associated gene, it is pattern of the aSSOciated also pOSSible possible that chromatin conformation and precise spatial arrangements between promoters and enhancers are important for the correct FIGURE 3. Expression of the SM22-1acZ SM22-lacZ transgene. The 1343 bp promoter of the temporospatial expression patterns SM22a gene was linked to lacz lacZ and used to create transgenic mice46 . (a) Whole mount of muscle-specific of genes. Thus, when a 12.5-day-old 12';-clay-old embryo showing shOWing SM22a-lacZ expression within the developing promoters and enhancers are sepavasculature, heart and somite myotomes (M). (b) Expression within the heart at this from their surrounding serated time point shows compartmentalized expression with high levels of SM22a-lacZ (A). quences and their orientations are expression in the right ventricle (RV) but not within the left ventricle (LV) or atria (A). SM22a-lacZ expression is also strong within the dorsal aorta (DA). (c, d) Transverse altered relative to those in the native sections through the esophagus (E) and dorsal aorta (DA) clearly show the specificity of gene, reproducible repnxludble alterations in their this transcriptional control module for arterial, but not visceral, smooth muscle. muscle. transcriptional specificity could result.
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Evolution of muscle cell diversity through modular enhancers
11 Ticho, B.S., Stainer, Y.R., Fishman, M.e. and Breitbart, R.E. (19%) Mecb. Dev. 59, 205-2]8 12 Lilly, B. et al. (1995) Science 267, 688-693 13 Bour, B.A. el al. (1995) Ge11es Dev. 9, 730-74] 14 Ranganayakulu, G. et al. (]995) Dev. BioI. 171. 169-181 15 Molkentin, ].D., Black, B.L., Martin, J.F. and Olson, E.N. (1995) Ce1l83, 1125-1136
Modularity of gene regulation in which multiple independent regulatory regions are required to generate the complete expression pattern of a gene during embryonic development and adulthood is not unique to muscle cells and has been carefully documented for numerous other types of genes in diverse organisms, particularly in Drosophila and sea urchin embryos (reviewed in Ref. 1). Why have muscle genes evolved SUcil complex sets of independent regulatory modules rather than having single enhancers for the control of expression in all muscle cells of a particular type? One possibility is that this type of modular regulation of gene expression provides greater flexibility to transcriptional control than might be achieved through a single regulatory module. In addition, modularity of transcriptional control sequences facilitates the establishment of complex patterns of gene transcription during evolution. Thus, in a simple organism with a relatively low complexity of muscle types, a single regulatory module might be sufficient to direct the complete expression pattern of a muscle gene, whereas the activation of muscle gene expression in more complex organisms could be achieved by the duplication of modules and concomitant recruitment of additional regulatory elements that confer positional specifiCity. The dependence of myogenic regulatory modules on combinations of cis elements makes it possible to create novel pat1erns of gene expression by combining different regulatory elements. As more myogenic regulatolY modules are identified and their regulatory factors are characterized, it should be possible to design myogenic regulatory modules that confer precise patterns of temporospatial regulation of muscle gene expression by combining regulatolY factor binding sites. 'J11is will then provide the opportunity to express exogenolls genes se)e<.1ively in different types of muscle cells ~Jt specific limes in devclopmcnt.
16 Kaushal, S., Schneider, J.W., Nadal-Ginard, B. and Mahdavi, Y. (1994) Science266, 1236-]24.0 17 Lassal', A.B. et al. (1989) Ce1/58, 823-831 Mol. Cell. Bioi. 13, 2753-2764
20 Fabre-Suver, C. and Hauschka, S.D. (996) J. BioI. Cbem. 271,4646-4652
O;erjesi, P., Ully, B., Hinkley, C., Perry, M. and Olson, E.N. (1994) J. BioI. Cbem. 269, 16740-]6745 22 Donoghue, M]. and Sanes, J.R. (] 994) Trends Ge1tel. 10,
21
396-401
(1993) DeIJelopmenl118, 61-69 24 Braun, T. et al. (1994) Development 120,3083-3092 25 Patapoutian, A. et al. (l995) Development ]21,3347-3358
26 Olson, E.N., Arnold, Ii.H., Rigby, P.W. and Wold, BJ. (]996) Cel/85, 1-4
27 Asakura, A., Lyons. G.E. and Tapscott, S.J. (]99,) Dev. BioI. 17], 386-398
Faennan, A. and Shani, M. (]993) DevelojJment 118, 919-929 29 Goldhamer, DJ. et al. (995) Development] 2], 637-649 30 Goldhamer, DJ., Faetman, A., Shani, M. and Emerson. e.P., Jr (}992) Science 256, 538-542 31 Hughes, S.M. et al. (993) DelJelojJme1t1 118, 1137-1147 32 Cheng, T.e., Wallace, M.e., Mel'lie, J.P. and Olson, E.N.
28
(993) Science 26], 215-28
33 Vee, S.P. and Rigby, P.W. (]993) Genes Det). 7, 12n-1289 34
7 Patapollti:l11, A., \'(/old. B.J. and \X':lgner. itA. (]99S) Sc;iemce270. ]H]H-JH2]
8
Donoghue, M. el al. (]988) Genes Dev. 2, ]n9-1790
35 Grieshammer, u., Sassoon, D. and Rosenthal, N. (1992) Ce1l69, 79-93 36 Donoghue. M.J. et al. (992) Ce1l69, 67-77
37 Rao, M.\l., Donoghuc, M.J., Merlie, .J.P. and Sanes, J.R. (1996) Mol. Cell. BioI. 16,3909-3922
38 McGrew. M.J. et al. (J996) Mol. Cell. BioI. ]6, 4524-4534 39 Kclly, R elal. (995)]. Cell. BioI. 129, 383-396
We thank members of our laboratory for helpful COI11ments, and 11. Tii'.enor (lnd 11. Hawlf for assi~1~mcc with gr'dphic'i. Work in our laboratory ill supported by gmntll from 'J11c NatjonaJ Institut.es of Health, 'J11e American He.lIi Associclliot1, ')11e Hohe!1 A. Wekh Foundation, the HlIll1~lJ1 Frontiers Sciences Program
Patapoutian, A., Miner, J.H., Lyons, G.E. and Wold, B.
23
Acknowledgements
References
Mueller, P.R. and Wold. B. (]989) SCience 246, 780-786 Amacher, S.L., Buskin, J.N. and Hauschka, S.D. (993)
18 19
40 Banerjec-Basu, S. and Buonanno, A. (1993) Mol. Cel/. BioI. 13, 7019-7028 41 Shield, M.A., Haugen, H.S., Clegg, e.H. and Hauschka, S.D. 09%) Mol. Cel/. BioI. ]6,5058-5068 42 Spitz, F., Salminen, M., Dcmingnon, J" Kahn, A., Daegelen, D. and Maire, P. (997) Mol. Cell. 13;01. 17. 656-666 . 43 Kuisk, J.Jt, U, H., Tran, D. and Capetanaki, Y. (]996) De". BioI. 174. ]-]3 44 Li. Z., March:md. P., Humbert, J., Babinel, C. and Pat!lin. D. (993) J)(!tJelojJm(Jl111 ]7, 947-959 45 Bihen, e., Hadchouel,,1., Tajhakhsh, S. and Bllckingham, M. (J996) Det). BioI. 173, 200-2]2 46 Li. 1.., Miano,,1.M., Mercer, B. and Olson, E.N. (996) .f. Cel/. BioI. 132, H49-859 47 Kim, S. el al. (997) Mol. Cel/. BioI. 17.2266-2278 48 SrivtlSI:1V:I, D., C'\crjcsi, P. and Olson. E.N. (995) Sciel1ce 270, ]995-]999 49 Srivastava, D., 11101113S, T., Un, Q. and Olson. E.N. Nal. G(!11el. (in press) . . 50 Li. I.. el al. (]997) Dell. BioI. ]87, 31 ]-32] 51 Olson. E.N. and Hosenthal, N. (J994) Ce1l79, 9-]2
"ossell, l..A., KelVin, 0 ..1., Sternberg. E.A. and Olson. E.N. (JI)H9) Mol. O!//. mol. 1),5022-5033
9 Olson, E.N., Perry. M. and S('hlllz. itA. (1995) Dlm. BioI. ]72,2-11 10 Eclmondson, I)'(~" Lyons. ('.E., M:111in. ,l.F. mltl Olson. E.N. (]CJJ.i) /)(JwlojJl1wnl ]20.125]-]263
A.n. Ff.,."llf flIt-dE.N. 01.~o" am i11. tbe JJepfll1mm1.f of /v1oleculm' Biology and Oncology,Hamon CelU(!1'for Ba.";c CanceI'Rese(.l1·cb, 71le 1111itX!/'si{1' ofT(J:X:os Sotllbweslem Medical Cel11(Jl'af Dallas, Dallas, TX 75235-9148, USA.
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