THE JOURNAL OF BIOLM~ICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 43, Issue of October 28, PP 27021-27028, 1994 Printed in U.S.A.

Combinatorial Structure of a Body Muscle-specific Transcriptional Enhancer in Caenorhabditis eZegans*

(Received for publication, May 31, 1994, and in revised form, July 29, 1994)

Verena Jantsch-Plunger$ and Andrew Fire$ From the Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210

We describe the dissection of a body muscle-specific enhancer sequence contained within the Caenorhabdi- t i s ekgans myosin heavy chain gene unc-54. A 90-base pair segment that was sufficient for both enhancer func- tion and tissue specificity was subjected to mutational analysis. Several separated sites within this region were required for activity; mutations in these sites led to dra- matic decreases in enhancer activity, while substitu- tions in the intervening regions had minimal effects on activity. The individual sites appear to function as semi- independent and partially interchangeable enhancer subelements, as seen by our ability to create functional enhancers by constructing novel multimers and combi- nations. Four different enhancer subelements (desig- nated 0, 1, 11, and 111) were identified in this way. Al- though partially interchangeable, some differences between these subelements were evident. In particular, concatamers of site I11 exhibited the highest levels of activity but had a broader tissue specificity than the intact enhancer, including both hypodermal and muscle tissue. The specificity of the intact enhancer thus re- flects a combinatorial function of the specificities of the constituent subelements.

The initial steps in differentiation of muscle cells involve the activation of a large family of genes encoding both contractile components and the machinery regulating contraction. Muscle is not a single uniform tissue type, but rather a group of tissues with the common property of contractility. This suggests the existence of several programs for muscle differentiation (Stock- dale, 1992).

Several candidates for factors with key roles in myogenic differentiation have been identified. The best characterized to date are the MyoD family of helix-loop-helix transcription fac- tors (for review, see Edmondson and Olson (1993)). These are capable of driving muscle-like differentiation when expressed in certain non-muscle tissue culture lines (see, e.g., Davis et al. (1987)). Genetic analysis in mice indicates a requirement for the MyoD family in normal muscle development (Hasty et al., 1993; Nabeshima et al., 1993; Rudnicki et al., 1993). Activation of genes encoding major muscle structural proteins could con- ceivably be carried out directly by MyoD family members or

* This work was supported by National Institutes of Health Grant R01-GM37706, by the Carnegie Institution of Washington, and by the Rita Allen Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must

U.S.C. Section 1734 solely to indicate this fact. therefore be hereby marked “advertisement” in accordance with 18

9 Submitted to fulfill part of the requirements for a doctorate of phi- losophy at the University of Vienna (Graduate Program in Biochemis- try). Present address: Austrian State Ministry of Health Research Labo- ratories, A-1031 Vienna, Austria.

$ To whom correspondence should be addressed: Dept. of Embryology, Carnegie Institution of Washington, 115 W. University Pkwy., Balti- more, MD 21210. “el.: 410-554-1234; Fax: 410-243-6311; E-mail: fire@maill.ciwemb.edu.

indirectly as a result of other transcription factors activated during myogenic commitment. In analyzing cis-acting se- quences responsible for the activity of muscle-specific promot- ers, several additional control elements have been identified that do not include MyoD target sequences (see, e.g., Gossett et al. (1988)). Searches for protein factors that interact with these sequences have led to the identification of the human myocyte- specific enhancer factor 2 protein family MEF-2 (Yu et al., 1992) and a mesodermally restricted homeodomain protein, MHox (Cserjesi et al., 1992).

Detailed analyses of muscle-specific control elements in ver- tebrate system have been carried out primarily by assaying activity in a set of myogenic and non-myogenic tissue culture cell lines. Although these assays provide a rapid means to iden- tify and characterize sequence requirements, they yield at best incomplete information concerning contributions to the pattern of activity within the developing organism.

Caenorhabditis elegans has been a productive model system for studies of muscle differentiation and activity at an organ- ismal level (Epstein and Bernstein, 1992). C. elegans contains two major muscle cell classes: body wall muscles used for loco- motion and pharyngeal muscles used for feeding (Waterston, 1988). These muscles are also distinct in gene expression; in particular, different myosin heavy chain genes are expressed in the two types. Body muscles express the myo-3 and unc-54 myosin heavy chain products, while pharyngeal muscles ex- press the myo-1 and myo-2 isoforms (Epstein et al., 1974). C. elegans can be readily transformed by microinjecting DNA

into oocytes or the syncytial germ line of adult hermaphrodites (Fire, 1986; Mello et al., 1991). The injected DNA is expressed in a large number of F1 progeny. The Escherichia coli gene lac2 serves as a useful reporter segment, allowing the pattern of expression for an injected transgene to be scored simulta- neously in all somatic tissues of transiently transformed F1 animals (Fire et al., 1990).

Using a combination of lac2 fusions and mutant rescue as- says, we have shown that each of the myosin heavy chain genes contains multiple regulatory regions, each of which is sufficient for activation in the appropriate muscle type (Okkema et al., 1993). unc-54, the most extensively analyzed of the four genes, contains an upstream activation site 120-160 bp’ 5’ t o the transcriptional start and an enhancer in the third intron. Ei- ther of these two elements is sufficient to direct unc-54 pro- moter expression in body muscles. The enhancer exhibits the stronger activation and can function in a position-independent manner relative to the promoter. We have therefore chosen the enhancer as the focus of fine structure analysis to determine the nature of sequences leading to gene activation in body muscles.

MATERIALS AND METHODS DNA Manipulations-Wild type and mutant 90-mer segments were

produced using four complementary oligonucleotides that anneal to

The abbreviation used is: bp, base paifis).

27021

27022 Modular Structure of a Nematode Body Muscle Enhancer

TABLE I A 90-bp segment of unc-54 functions as a body muscle specific transcriptional enhancer

Constructs pPD18.56, pOK11.34, and pPD26.50 are promoter::lacZ fusions that are (by themselves) inactive in body muscles. The 90-mer is a 90-base pair region with end points delimited by our initial analyses of enhancer activity (Okkema et al., 1993); the 90-mer is completely contained in the third intron of unc-54. MC93, MC240, and MC149 have a single copy of the 90-mer cloned (in a minus orientation) into restriction sites just upstream of the promoter segment in each of the parent vectors. MC176 has three copies of the 90-mer cloned in a tandem array upstream of the glp-1 promoter in pPD26.50. The “Activity” shown for each construct describes the staining pattern seen in F1 animals after germline injection of 10-12 parental adults. glp-1 and Amyo-2 basal activities generally gave 3-10 staining cells/injection set. Strong activity in body or pharyngeal muscles was evidenced by staining in 50-300 animals; positive animals generally show activity in many cells of a given muscle class. “Weak body muscle activity” for MC176 indicates staining in about 20 muscle cells of F1 animals derived from 12 injected hermaphrodites.

Construct Description

pPD18.56

pOK11.34

pPD26.50

MC93

MC240

MC149 MC176

myo-2::lacZ translational fusion (1172 bp up- stream + 909 bp transcribed sequence)

Deleted myo-2 promoter fused to lac2 (tran- scriptional fusion with 158 bp of upstream sequence from myo-2)

upstream sequence +25 bp coding sequence) glp-1::lacZ translational fusion (2512 bp of

90-mer upstream of pPD18.56

90-mer upstream of pOK11.34

90-mer upstream of pPD26.50 3 x 90-mer upstream of pPD26.50

Activity

Strong activity in pharynx muscles

Amyo-2 basal activity (very rare staining in pharyngeal muscles and marginal cells)

glp-1 basal activity (rare hypodermal stain,

Strong activity in body muscles (+ strong

Strong activity in body muscles (+ Amyo-2

particularly hyp-10; very rare pharyngeal stain)

activity in pharynx muscles)

basal activity) glp-1 basal activity Weak body muscle activity (+ glp-1 basal

activity)

produce a 90-bp fragment that can then be inserted directionally into unique upstream restriction sites in pPD18.56. This procedure was chosen over standard in vitro mutagenesis procedures to provide greater mutational flexibility; a disadvantage was that at least two different oligonucleotides (both strands of the relevant region) must be synthesized for each mutant enhancer. All mutated enhancers were sequenced to verify that no unexpected mutations had been introduced.

Mutations were designed to yield a novel substitution with minimal homology to the wild type sequence. In addition, several of the sequence substitutions result in the formation of novel restriction sites. These sites were subsequently used to generate combinations and duplica- tions of different 90-mer elements, as well as short insertions and deletions. To construct concatamers of individual sites, sense and antisense

oligonucleotides were phosphorylated with T4 polynucleotide kinase, boiled, cooled to 23 “C, and treated with T4 DNA ligase (Vinson et al., 1988). Concatamers of the desired size were then gel-purified.

The lac2 fusion constructs used as starting points for enhancer as- says (pPD18.56, pPD26.50, and pOK5.56) have been described (Okkema et al., 1993); pOK11.34 is equivalent to pOK5.56 with a set of cloning sites added upstream of the deleted myo-2 promoter.

Microinjection, Histochemical Staining, and Activity Determin- ation-Plasmid DNA prepared as described (Fire et al., 1990) was in- jected into gonadal syncytia of 5-12 wild type hermaphrodite adults (Mello et al., 1991). Injected animals were allowed to self-fertilize at 20 “C. After 4 days, animals were harvested in H,O, lyophilized, fured with acetone, and stained to localize p-galactosidase activity (Fire, 1992). Stained animals were examined under a dissecting microscope (magnification, x25-50) and under compound optics (magnification, x200400), with the latter being necessary to detect faint staining.

RESULTS

Assays for Enhancer Function in Body Muscle-We have pre- viously described a sensitive assay to identify enhancer seg- ments active in body muscles (Okkema et al., 1993). This assay is based on the high frequency of DNA transformation observed in the first generation following microinjection into C. elegans germ line (Mello et al., 1991). Because these F1 animals are in general mosaic for the transforming DNA, complete expression patterns are actually deduced by examining expressing tissues in a population of 50-300 transformed F1 animals. The starting point in our initial assays for body wall muscle enhancer func- tion has been a myo-2::lacZ fusion construct (pPD18.56) that is expressed exclusively in pharyngeal muscles. We test enhancer function by placing specific fragments upstream of the myo-2 promoter in this construct. Enhancement in body muscle is

readily scored; we identified five distinct body muscle enhancer elements associated with the genes unc-54 and myo-3 (Okkema et al., 1993).

Although the pPD18.56 construct provides a sensitive assay to identify enhancers active in body muscle, it gives only a limited view of tissue specificity for a given enhancer element. High level expression of pPD18.56 in pharyngeal muscles pre- vents us from scoring enhancement in any pharyngeal tissue. In addition, the myo-2 segment in pPD18.56 shows a bias in ability to respond to enhancement, responding primarily in muscle tissue (Okkema et al. 1993).

Two different promoter segments were used to obtain a more complete view of tissue specificity for individual enhancer seg- ments. A “minimal promoter” fusion derived from myo-2 (pOK11.34) gives essentially no activity alone but can respond to enhancement in body muscle, hypodermis, pharyngeal mus- cle, and other pharyngeal tissues (Okkema et al. 1993; this work; see Footnote 212 Enhancement assays using this deleted myo-2 promoter segment have been particularly useful in ad- dressing the difference between enhancers with general muscle activity and those with activity limited to body muscles.

To test enhancer tissue specificity in a less restrictive assay, we needed a canonical promoter with no muscle specificity. For this purpose, we used the upstream sequence from the gene glp-1 (Yochem and Greenwald, 1989). The endogenous glp-1 expression pattern has not yet been fully characterized, but genetic data suggest a role in multiple tissues including germ line (Lambie and Kimble, 1991). Our canonical glp-1::lac.Z fu- sion (pPD26.50) contains just sequences upstream of the gene and shows only low levels of background staining, primarily in a subset of hypodermal cells in the tail (Okkema et al., 1993). This glp-1 promoter segment can respond to enhancement in body wall muscle; placing a 963-bp fragment of unc-54 internal sequence upstream of the glp-1 promoter results in reproduci- ble body wall muscle expression (Okkema et al., 1993). Other experiments have shown that the glp-1 promoter can respond to enhancer function in pharyngeal, neural, glial, and hypoder- mal tissue (this work; see Footnote 313 We therefore take the

S. Xu and A. Fire, unpublished data. P. Okkema, S. White Harrison, S. Xu, I. Johnstone, personal com-

munication.

Modular Structure of a Nematode Body Muscle Enhancer

A* construct

MC126

MC107 MC210 MC223

MC138 MC140

MC270

MClOl

B !TCCCATTCTATCATCAATTt!GTGGGATGAGGCTATCTCLGCCTCTCll $ K A A T C T C T $ P C C A T C l l !$ATTACACT 8G;rGGATGACG

I I I11

+(wk) 25

+ (wk) 26 +/-(wk) 17

+ (wk) 45 +/- (wk) 8

2

++ 89

0

+ (wk) 60 2 0 0

2

+ (wk) 39

++ 90 ++ 88

++ 85

1 0 0

+ (wk) 40

++ 72

++ 89

enhancer sequence (present in construct MC93) is shown at the top. Each line below represents a single mutant construct. All changes from the FIG. 1. Mutational analysis of the 90-mer enhancer. A, mutant 90-mer sequences assayed for enhancer activity. The wild type 90-mer

original 90-mer sequence are noted, while a horizontal line indicates sequence identical to the original. The constructs shown contain the mutated enhancer sequences (single-copy, inverted orientation) upstream of the complete myo-2::ZacZ fusion pPD18.56. Each construct was strongly active in pharyngeal muscles as a result of the myo-2 promoter. Activities in body wall muscle are indicated by strength and frequency of staining. The

were assigned based on this frequency as follows: +++ (90-100%), ++ (60-90%), + (20-60%), f (5-20%), and - (<2%). Intensity of staining could not frequency (fur right) is expressed as the percentage of animals with control staining in the pharynx that also show body muscle staining. Activities

be precisely quantitated, but consistently weak staining was observed for a subset of constructs. Constructs with body wall activity scored as ++(wk), +fwk), or d w k ) gave faint blue staining that was not evident in the dissecting microscope but was visible using more sensitive compound (brightfield) optics; constructs scored as +++, ++, or + all showed strong body muscle staining visible in both dissecting and compound brightfield microscope (Fire, 1992). As an internal control, pharyngeal staining, visible with either dissecting or compound optics, was similarly intense with all constructs tested. B , summary of 90-mer mutational analysis. Summary of data in panel A. Substituted regions are indicted by burs: white (enhancer activity a t levels comparable to the wild type gO-mer), gray (dramatic decrease in enhancer activity), and black (elimination of enhancer activity). In general, consistent results were obtained in regions covered by more than one substitution. A slight anomaly in the site I1 region is indicated by that weak staining in body wall muscle of construct MClOl (which contains an 11-bp substitution), while shorter substitutions entirely contained within the 11-bp region (MC140 and MC270) were completely inactive. Although we have no direct explanation for the anomaly, it seems likely that absolute level of activity for these site I1 substitutions depends on the precise sequence introduced by the substitutions.

pattern of induced expression from the glp-1 promoter segment fragment contained in the unc-54 third intron can activate body as an indication of enhancer tissue specificity in the context of wall muscle expression when placed in either orientation up- a naive promoter. stream of the myo-2 promoter in pPD18.56. We refer to this

A 90-base Pair Segment within the Third Intron of unc- 90-bp restriction fragment as the "90-mer." The tissue specific- 54 Functions Independently as a Body Muscle-specific ity of enhancement directed by the 90-mer was investigated by Enhancer-We had shown previously that a 90-bp restriction assaying its function in different promoter contexts (Table I). A

27024 Modular Structure of a Nematode Body Muscle Enhancer

single copy of the 90-mer functions as a strong enhancer up- stream of the deleted myo-2 promoter segment in pOK11.34. The observed enhancement occurs solely in body muscles; no enhancement is seen in pharyngeal muscles or in other tissues.

Enhancement of the glp-1 promoter by the 90-mer is some- what less pronounced. A single copy of the 90-mer was insuffi- cient to produce enhancement of the glp-1 promoter in pPD26.50. However, when we placed three copies of the 90-mer in tandem upstream of pPD26.50, we observed low level en- hancement, which occurred specifically in body wall muscles. The low activity of the 90-mer enhancer linked to the glp-1 promoter might reflect an added level of sequence or conforma- tional complexity in the generation of an efficient promoter or enhancer interaction. Nonetheless, the specificity of enhanced expression from both the glp-1 and deleted myo-2 promoters suggested that the 90-mer sequence carries sufficient sequence information to act independently as a body muscle-specific en- hancer element. Given the sufficiency of the 90-mer segment for tissue-specific enhancer function, we have chosen to focus on this element in initiating a more detailed mutational analysis.

Multiple Distinct Regions within the 90-mer Are Required for Enhancer Activity-A series of mutated 90-mer sequences were tested for enhancer activity upstream of the myo-2::lacZ fusion pPD18.56. Because these constructs were derived from pPD18.56, all showed strong expression in pharyngeal muscle cells. The pharyngeal staining was used as a control for the quality of transformation. At least 120 staining animals for each construct were scored for additional staining in body mus- cle cells. Fig. lA summarizes the results of this analysis. High levels of enhancer activity resulted in strong staining in body muscle cells. For fully active constructs, 70-90% of the animals that showed control staining in pharyngeal muscle cells also stained in body muscle cells. Mutations that decreased en- hancer activity generally caused a decrease in both frequency and intensity of body wall muscle staining. Fig. 2 shows ex- amples of stained F1 animals resulting from injections of con- structs from the different classes.

The mutational analysis indicated that sequences critical for activity are distributed across the length of the 90-mer en- hancer. Mutations in regions 1-32,37-50, and 75-85 (sequence shown in Fig. LA) each led to dramatic decreases in enhancer activity. By contrast, substitutions in between these regions did not affect the activity of the enhancer. This analysis led to the hypothesis (Fig. 1B) that this enhancer contains several essen- tial sites, which we named 04, 11, and 111. (Although the mu- tational analysis in Fig. L4 does not distinguish two sites in the 1-32 region, subsequent experiments described below led us to divide the region into two sites, which we designate 0 and I.)

To investigate the nature of interactions between the four essential sites in the enhancer, we have tested the effect of altered spacing between these regions (Fig. 3). Insertion or deletion of a few base pairs between sites I and I1 or between sites I1 and I11 substantially decreased or eliminated enhancer activity. This indicated that phasing of these adjacent sites was important for proper enhancer function. An alternative means for altering spacing within the enhancer was used in MC152 (Table 11); in this construct, the spacing between sites located at the 5' end (0 and I) and the 3' end (111) was increased by duplicating site 11. MC152 showed strong enhancer activity (in fact exhibiting somewhat more activity than the original 90- mer construct). The strong activity of MC152 indicated some flexibility in assembling functional activation complexes at the site of the enhancer.

Duplication of Inactive Mutant Enhancers Can Restore Actiuity-The results with MC152 caused us to investigate the

tant 90-mer segments. Animals shown are the F1 progeny of adult FIG. 2. Activation of myo-2lacZ fusions by wild type and mu-

hermaphrodites injected with DNAconstructs shown in Fig. L4 and are stained with 5-bromo-4-chloro-3-indoyl p-D-galactoside. Each construct represents a wild type or mutant 90-mer segment upstream of the complete myo-2::lacZ fusion pPD18.56. PaneZA, MC93 (++), fully active enhancer segment; strong body wall and pharyngeal staining. Panel B, MC175 (+(I&), weak enhancer; faint and relatively rare body wall staining (with strong control staining in pharynx). Panel C , MC97 (-), inactive enhancer; only pharyngeal staining is observed. Staining in these samples is both cytoplasmic and nuclear; an SV40 nuclear local- ization signal from the vector at the junction of myo-2 and lac2 se- quences in pPD18.56 (Okkema et al., 1993) is partially masked, leading to this mixed localization.

possibility that alternative combinations of the four sites might be functional as enhancers. As an initial test of this, we con- structed duplications of mutant enhancers that were non-func- tional in single-copy due to substitutions in sites I, 11, or I11 (Table 11). In each case, enhancer activity was restored by the duplication. The duplicated site I1 mutation is a particularly

Modular Structure of a Nematode Body Muscle Enhancer 27025

FIG. 3. Effects of altered spacing on enhancer activity. Activities and struc- tures of constructs are described as in Fig. 1. For each construct, the net alteration in spacing is given (e.g. +2, -4). Constructs MC370 and MC154 display very weak staining in body wall muscles, although frequency measurements are comparable to the wild type enhancer.

construct

MC93 ATCCCATTCT CTCATCAATT GAGTGGGATG AGGCTATCTC :!CCTCTCTT CTGAATCTCT GAACCATCTT ACATTACACT GTGGATGACG ++ 87 11 21 3 , 51 61 7 1 81

GAATGAAGG

MC157 ATCCCATTCT CTCATCAATT GAGTGGGATG AGGCTATCTC TGCCTCTCTT CTGd -' L A T C T T ACATTACACT GTGGATGACG ++ (wk) 85

<MTGC$4GCAA?

MCl2l ATCCCATTCT CTCATCAATT GAGTGGGATG AGGCTATCTC TGCCTCTCTT CTGA ATCTT ACATTACACT GTGGATGACG - n

TABLE I1 Duplication can restore activity of inactive enhancer mutations

MC104, MC107, and MC97 and measurement of activity levels are as described in Fig. 1. Enhancer segments shown here were tested upstream of the complete myo-2 promoter (parent construct pPD18.56). Monomer constructs MC93,

Construct Description promoter (pPD18.56) in body muscle Activation of the complete myo-2

MC93 Wild-type 90-mer (0 + I + I1 + 111) MC152 (0 + I + I1 + I1 + 111) MC104 MC147 MC107 MC206 MC97 MC189

Site I substitution (0 + [il + I1 + 111) Duplicated MC104 (0 + [il + I1 + III), Site I1 substitution (0 + I + [iil + 111) Duplicated MC107 (0 + I + [iil + HI), Site 111 deletion (0 + I + 11) Duplicated MC97 (0 + I + II),

TAEILE I11 Oligonucleotides used to generate subelement multimers

Only plus strand oligonucleotides are shown. Uppercase letters de- note bases derived from unc-54 enhancer sequence; lowercase letters denote other sequences (5' linkers and substitution mutations). V P O includes the first 13 bases of the enhancer; W I , W I I , and VPIII cover bases 13-31, 35-52, and 64-88, respectively. Complementary minus strand oligonucleotides for each oligonucleotide described above were synthesized to generate double strand segments with 5' overhang se- quences of caag and cttg at the two ends. This allows facile and direc- tional cloning of multimers into an asymmetric Sty1 site (C/CTTGG).

Oligonucleotide Sequence

V P O CaagGTCTCTATCCCATTCTCTC VPI CaagCATCAATTGAGTGGGATGA VPII CaaggTATCTCTGCCTCTCTTCT

W I * CaagGAagttaactGTGGGATGA vPIII"6 CaaggCCATCTTACATTtACTGTGGATGA VPIII"2 CaaggCCATCTTACATTACAgacaGGATGA

VPIII CaaggCCATCTTACATTACACTGTGGATGA

__

good example. As a monomer, the site 11 mutation MC107 showed no activity; a duplication of this region (MC206) gives an enhancer that is actually more active than the original wild type 90-mer. Similar results were observed in duplicating the site I substitution (monomer, MC104 (2); dimer, MC147 (+++)), while duplication of the site I11 substitution resulted in only partial restoration of enhancer activity (monomer, MC97 (-); dimer, MC189 (+wk, where wk indicates weak staining)). These results indicate that defects in critical regions of the enhancer can be compensated by reiteration of the other sites; this sug- gests in turn that these sites are at least partially interchange- able.

The unc-54 Enhancer Contains at Least Four Functional Subelements-The above data are consistent with the hypoth- esis that a functional enhancer requires a minimal number of

++ +++

+/-(wk) +++ -

+++

%

87 96 5 96 0 96

- 0 + (wk) 12

TABLE N Activation of the complete myo-2 promoter by multimerized

enhancer subelements Multimers of individual subenhancer elements from Table 111 were

assayed for activity upstream of the complete myo-2 promoter (~PD18.56). Activity levels are Dresented as in Fie. 1.

Construct Description plete myo-2 promoter in Activation of the com-

body muscle

MC388 (I), MC391 (II), MC393 (III), MC395 (19,

%

+(wk) 20 0

+++ 80-100 0

-

- MC194 I1 + 111 - 0 MC322 MC213 I1 + I11 + (I), +++ 100 MC319 I1 + 111 + (I#), - 0 MC217 MC265

84

I1 + 111 + (O), + 53

I1 + I11 + (II), ++ I1 + I11 + (III), +++ 100

enhancer subelements in a properly phased array. To test this model, we synthesized oligonucleotides corresponding to each of the individual sites identified from the mutational analysis (Table 111). The optimal phasing between individual sites was not intrinsically evident from our earlier analyses, but a rea- sonable start was to use concatenates in which individual sites were separated by an integral number of helical turns (see, e.g., Hardy and Shenk (1989) and Carra and Schleif (1993)). It should be noted that the activities for these constructs would reflect both the activities of the individual elements present and their precise arrangement in the reconstructed enhancer; thus, low activity might reflect either a weak combination of enhancer subelements or non-optimal spacing between ele- ments. In any case, a positive result (activity of a construct in

27026 Modular Structure of a Nematode Body Muscle Enhancer

TABLE V Enhancement of the deleted myo-2 promoter and glp-1 promoter by multimerized enhancer subelements

segment (pPD26.50). These constructs all exhibit a low level of background activity (indicated by (-)) corresponding to the basal activity of the Multimer of individual enhancer subelements (Table 111) were assayed upstream of the deleted myo-2 promoter (pOK11.34) and aglp-1 promoter

promoter used. Additional staining reported for each of the constructs is distinct from this background, with the possible exception of the rare hypodermal staining seen with MC543 and MC551.

Enhancer elements

Activation of the deleted myo-2 promoter (pOK11.34) Activation of the glp-1 promoter (pPD26.50)

Construct Staining pattern Construct Staining pattern

(O), MC460 (-1 ND (I), MC379 Very weak body muscle MC397 (I#),, MC384 (-) MC401 (111, MC382 (-1 MC399 (III), MC383 Strong body muscle, MC400 Body muscle, hypodermis

I1 + I11 + (01, ND MC350 Weak body muscle I1 + 111 + (I), MC355 Weak body muscle MC280 Weak body muscle I1 + 111 + (I#), MC362 (-) MC347 I1 + I11 + (IO4 MC356 Weak body muscle I1 + 111 + (1111, MC358 Strong body muscle MC284 Body muscle, hypodermis (111196 MC524 (-1 MC543 Rare hypodermis (faint

(111194 MC331 (-1 MC551 Rare hypodermis (faint

(-) (-1 (-1

weak hypodermis

(-1 (-) MC282

stain, mostly in head)

stain, mostly in head)

body wall muscles) is an indication that we have created a transcriptional enhancer that acts in those cells.

Concatamers of each individual site were initially tested up- stream of the complete myo-2 promoter in pPD18.56 (Table IV). In this context, a site I11 concatamer gave strong enhancement in body wall muscles while a site I concatamer exhibited weak enhancer activity. No activity was observed with site 11.

To provide a more sensitive assay for function of the con- catamerized enhancer elements, we carried out similar assays using a “sensitized” promoter construct that already contained copies of sites I1 and I11 (MC194). This construct was inactive by itself in body wall muscle but evidently provides a sensitive assay for functional enhancer subelements; the addition of mul- timers of any of the four enhancer subelements induced expres- sion in body wall muscle. Enhancement was strongest in the case of site I11 and progressively weaker for sites I, 11, and 0. As a control to show that enhancement in these constructs was not a result of linker sequences or concatamer structure, a mutated site I oligonucleotide (I#) was concatenated and failed to show any enhancement activity.

Overlapping Tissue Specificities of Enhancer Sub- elements-To this point, we had used the complete myo-2 pro- moter (i.e. pPD18.56) in dissections of the functional enhancer. This assay primarily measures enhancement in body wall muscles (Okkema et al., 1993). Assays using combinations of the individual sites upstream of the glp-1 and deleted myo-2 promoters give a more general indication of tissue specificity of enhancer function; these are presented in Table V. When the individual concatenated sites were tested for their ability to activate the minimal myo-2 promoter or the glp-1 promoter, only site 111 showed very high activity, while sites 0, I, and I1 showed only low levels of body muscle activation (in some cases requiring “sensitized” promoter constructs as described above).

The glp-1 constructs containing multiple copies of site I11 exhibited a surprising tissue distribution, showing activity in hypodermal cells as well as body muscles (Table V, Fig. 4). Hypodermal enhancement by the multimerized site I11 ele- ments was also observed using the deleted myo-2 promoter (although at lower levels than with the glp-1 promoter).

The broadened specificity of site I11 activation in the glp-1 promoter context could reflect several conditions. In particular, site 111 could bind a single trans-acting factor functioning both in body wall muscle and hypodermal cells or two different fac- tors with distinct hypodermal and muscle tissue specificities.

To address this question, we assayed hypodermal and body muscle activities of several variants of site 111. These mutated sites were designed based on the linker scan mutagenesis; each mutant oligonucleotide pair corresponds to a change around site I11 that renders the 90-mer inactive in body wall muscle. Each oligonucleotide was concatamerized and tested for activ- ity both with the deleted myo-2 promoter (MC524 and 531, Table V) and theglp-1 promoter (MC543 and MC551, Table V). In either promoter context, the mutated constructs show no body wall muscle expression. With the glp-1 promoter, a low level of hypodermal cells staining was observed, predominantly in the head. This expression appeared less extensive than we had observed with the non-mutant site I11 multimers. This suggests that hypodermal and muscle activities of site I11 are driven by factors that bind similar or identical target sequences.

DISCUSSION We have described a detailed analysis of a body muscle-

specific enhancer sequence responsible for activation of the unc-54 major myosin heavy chain gene. A 90-base segment sufficient for enhancer function was extensively mutagenized and shown to contain four essential sites, referred to as 0, I, 11, and 111. Mutations replacing sequences in any of these four sites eliminated or dramatically reduced activity of the en- hancer element. In contrast, the regions between sites I and I1 or between sites I1 and I11 could be replaced with unrelated sequences without affecting enhancer function. The spacing between sites within the enhancer appears to be important; short insertions and deletions in nonessential interspersed se- quences lead to decreases in enhancer activity. Presumably this reflects a closely interacting complex of proteins occupying this enhancer.

To further dissect the enhancer segment, we needed an assay to characterize individual sites in a context independent of the full enhancer. Single sites were concatamerized and tested for activation of various promoters. Each of the four enhancer sub- elements showed some activity in these assays, although con- catamers of site 111 showed by far the strongest enhancement. Multimers of this subelement alone were sufficient to activate any of the promoters tested. Given the strong activity of the site I11 multimers in the assay, it might be of interest to ask why the unc-54 gene contains a more complex combination of sites in- stead of a functional site I11 multimer. Although the multimer-

Modular Structure of a Nematode Body Muscle Enhancer 27027

FIG. 4. Hypodermal and muscle activation of glp-1 and deleted myo-2 promoters by site 111. Animals shown are F1 progeny of adult hermaphrodites injected with DNA constructs shown in Table V. Each construct represents a concatamer of enhancer subelement I11 (four tandem copies) upstream of a minimally active lac2 fusion. 5-Bromo-4- chloro-3-indoyl P-D-galactoside staining in these samples is fully nu- clear, as a result of an unmasked SV40 nuclear localization signal appended to lac2 in the parent vectors (Fire et al., 1990). Identity of stained cells was confirmed by comparison with the overall nuclear pattern, visualized by simultaneous staining with the fluorescent dye 4,6-diamidino-2-phenylindole (not shown). Panel A, body wall muscle staining with MC383.site 111 concatamer upstream of deleted myo-2 promoter. Panel B, hypodermal (lower cells) and muscle (upper cells) staining with MC400,site 111 concatamer upstream of glp-1 promoter. Panel C , body wall muscle staining with MC3584te I11 concatamer

ized site I11 exhibited activity comparable to the complete en- hancer, some loss of tissue specificity was evident. In particular, staining in hypodermal cells of the body was seen when this subelement was multimerized upstream of the glp-1 promoter. The precise tissue specificity of the intact enhancer results from the requirement that all subelements of the en- hancer function simultaneously.

Vertebrate studies implicate the MyoD family of helix-loop- helix motif DNA-binding proteins in regulation muscle gene expression. One C. elegans homologue of this family (called HLH-1) has been described (Krause et al., 1990). Although HLH-1 is expressed in body muscle precursors, analyses of genetic deficiencies demonstrated that the factor was not ab- solutely required for unc-54 expression (Chen et al., 1992). Sub- sequently, we have shown that the unc-54 enhancer and pro- moter can each function in hlh-1 mutant animals (ZacZ fusions driven by either the enhancer or promoter alone express fully in mutant background^).^

The analysis of hlh-1 mutations did not rule out the possi- bility that some other HLH factor could be responsible for unc-54 activation. The unc-54 enhancer contains a single po- tential HLH target site (“E box,” CAnnTG), which lies within enhancer subelement I (CAATTG). Detailed mutagenesis of this site revealed that enhancer activity does not require HLH or MyoD family consensus sequences (Fig. 1). In particular, mutations that eliminate the E box (MC172, gAATTG; MC193, CAATTc) were still partially functional as enhancers in vivo, while a mutation improving the MyoD consensus (MC175, CAgcTG) actually decreased enhancer activity.

Detailed sequence comparison between hlh-1 genes from sev- eral different nematode species reveals a set of highly con- served sites within the first intron of hlh-1 which are similar to the essential sites within the unc-54 enhancer (Krause et al., 1994). Although this conserved region is unlikely to account completely for the complex hlh-I expression pattern, it is in- triguing to consider that activation of unc-54 and maintenance of hlh-1 expression may involve similar sets of regulatory factors.

I t is likewise of interest to draw a comparison between the body muscle-specific unc-54 enhancer and the pharyngeal mus- cle-specific (myo-2) enhancer that has been characterized con- currently (Okkema and Fire, 1994). (Pharyngeal muscle is a non-striated muscle type that is distinct from the body muscu- lature (Epstein et al., 1974; Waterston, 1988).) For both the unc-54 and myo-2 enhancers, the active region combines sub- elements with distinct specificities. In the case of myo-2, an organ-specific signal active throughout the pharynx combines with signals that are limited to pharyngeal muscles. The prop- erties of the unc-54 enhancer appear similar in that a strong but more general region-specific signal (site 111, which is active in body wall tissues including both muscle and hypodermis) is restricted to muscle expression by requiring the function of adjacent elements. The nature of these organ- or region-specific control signals should illuminate the early decisions leading to pattern formation and organogenesis.

Our analysis of the unc-54 enhancer indicates that several different trans-acting factors have specific roles in activating gene expression in C. elegans body muscles. Some of these,

V. Jantsch-Plunger, L. Chen, and A. Fire, unpublished data.

upstream of sensitized myo-2 promoter. Panel D, hypodermal staining with MC284.site I11 concatamer upstream of sensitized glp-1 promoter. Photos in C and D are slightly out of the nuclear focal plane to show cell-type-specific features (arrows); filled arrow in C , muscle filaments; hollow arrow in D, alae structures (three parallel longitudinal folds in the cuticle characteristic of regions overlying hypodermal nuclei) (White, 1988).

27028 Modular Structure of a Nematode Body Muscle Enhancer

including the factor(s) responsible for interaction with site 111, may have a broader tissue specificity than simply body muscles. This is a consideration in designing genetic screens for identification of trans-acting factors; in particular, mutations affecting a factor with broadened tissue specificity might have pleotropic effects on non-muscle tissues that would initially prevent them from being isolated in screens for muscle defects (see, e.g., Williams and Waterston (1994)). The precise defini- tion of the cis-interacting sequences reported here should be a good starting point in biochemical and molecular identification of the factors responsible for trans activation (Vinson et al., 1988; Singh et al., 1988).

Acknowledgments-We thank J. Ahnn, L. Chen, L. Connah, I. John- stone, M. Krause, A. Pinder, P. Okkema, R. Schleif, s. Xu, W. Kelly, and G. Seydoux for help and suggestions over the course of this work. We thank W. Loffelhardt as external academic advisor for I? J.-P.

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