Expression profiles and unc-27 mutation rescue of the striated muscle type troponin I isoform-3 in Caenorhabditis elegans

Transcription control of multiple genes in tissueand stage-specific patterns is still of major interest. We show here that troponin I (TNI) is expressed under the control of upstream non-coding sequences and had functions as an isoform of intermediate type between pharynx and body-wall of the gene. In Caenorhabditis elegans, three striated muscle TNIs are expressed in body-wall muscles and a cardiac isoform is expressed in the pharynx. We have analyzed the gene expression mechanisms of tni-3 gene and motility function of its protein product. Promoter deletion analysis of the tni-3 gene identified muscle enhancers including the head enhancer. The CBF1/Su(H)/LAG-1-binding motif was included in the head enhancer. Yeast one-hybrid screening isolated the lag-1 clone in five candidates. Functional differences between the three striated muscle TNIs were investigated by the expression of promoter-fusion genes into tni-2/unc-27(e155) null mutant animals. The results suggest that the cis-elements in the promoters of the three genes are important for their tissue-specific expression and that from the function of TNI-3, the tni-3 gene would be an intermediate in the evolution of these genes by gene duplication. Mechanisms of tni-3 expression and its molecular function may contribute to our understanding of gene evolution and developmental programs.


INTRODUCTION
The troponin (TN) complex is composed of three polypeptides: troponin I (TNI), the inhibitory component, troponin C (TNC) that binds Ca 2+ and troponin T (TNT) that binds the whole complex to tropomyosin (TM)-binding (Ohtsuki et al., 1986).The TN complex is highly conserved in vertebrate and invertebrate, and in striated and non-striated muscles (Filatov et al., 1999;Nongthomba et al., 2004).The mechanisms controlling isoform expres-sion are of two types; one involves the upstream transcriptional control of each gene and the other is alternative splicing of the specific exons.In the nematode Caenorhabditis elegans, each of four TNI genes is expressed tissue-specifically by the control of each gene (Ruksana et al., 2005;Kagawa et al., 2007), whereas in Drosophila the TnI gene produces 10 isoforms by differential splicing of 13 exons, similarly to vertebrates (Barbas et al., 1993).
In C. elegans, there are two types of muscles; pharyngeal, corresponding to the heart, and body-wall corresponding to the striated skeletal muscles in vertebrates.Three TNI genes tni-1, tni-2/unc-27 and tni-3 are expressed in the body-wall muscles including those of the vulva and rectum muscles and a fourth, tni-4, is expressed only in the pharyngeal muscles (Ruksana et al., 2005).Different genes encode TNI-1 and TNI-2 but there is strong homology especially the identical amino acid sequences in their actin-binding site.Among the four TNI genes mutants are only known for the tni-2/unc-27 gene as unc-27(e155) null mutant animals.TNI-2, the most abundant troponin I isoform in C. elegans, is likely to play an important role in locomotion behavior involving in dynamic muscle contraction.In addition, the pharynx develops at an earlier stage than the bodywall muscles.Gene duplication is a main source of different genes and is a central factor influencing genome evolution (Lynch and Conery, 2000).We have proposed a gene duplication model of the four TNI genes arising from our analysis of the molecular interactions of their products with other TN components in vitro (Amin et al., 2007).Briefly, TNI-3 is a primitive body-wall type TNI that must be derived from the pharyngeal muscle type TNI-4 since it interacts with TNC from both muscle types.TNI-1 and TNI-2 are definitive body-wall types derived from TNI-3 as they only express in body-wall muscles and interact only with the body-wall type TNC.
Tissue-or stage-specific transcription factors binding to promoter cis-elements control gene expression during worm development and differentiation (Krause, 1995).Pharyngeal muscle gene expression is activated by NK-2/ CEH-22 homeoprotein (Okkema and Fire, 1994).The skeletal type of CeMyoD/HLH-1 regulates muscle gene expression in body-wall muscles (Chen et al., 1994), and CeTwist/HLH-8 and T-box factor MLS-1 regulate expression in vulval and uterine muscles (Kostas and Fire, 2002).We have reported that the expression profile of TNI-2 is similar to that TNI-1 at decreased levels (Ruksana et al., 2005).However, detailed molecular studies of the TNI-3 isoform expressing in body-wall muscles have not been reported.Completing all these studies for the three TNI isoforms will allow us to understand, not only their expression patterns but also their functional differences.
Here we studied the tissue-specific expression patterns of all three TNI isoforms using gfp and rfp reporters and their functional differences on motility by using transgenic animals.These were derived by injecting promoter/coding-chimera gene constructs from the three body-wall TNI genes into unc-27(e155) null mutant animals.The tissue-specific enhancer in the tni-3 promoter was determined and the regulatory proteins binding to the head-enhancer region of tni-3 was sought in vitro and in silico.

MATERIALS AND METHODS
Animal strain and culture All C. elegans strains used are derived from the wild-type Bristol strain N2 and were grown under standard conditions (Stiernagle, 1999).
Yeast one-hybrid screening Yeast one-hybrid screening was performed as described (Bando et al., 2005).Briefly, mixed staged C. elegans cDNA library (pACT-RB2, a generous gift from Dr. Robert Barstead) was transformed into yeast Saccharomyces cerevisiae YJL170 with the bait plasmid pHE3HIS (Supplementary Table S1) and was screened in the -Trp/-Leu/-His selection medium containing 3-amino-1,2,4-triazole (3-AT).Bait plasmid was generated by inserting the head enhancer sequence into pRS414/HIS (a generous gift from Dr. Peter Okkema).
Observation of animals and detection of transgenic gene products Each reporter fusion construct was microinjected into the wild-type N2 animals at 10-100 μg/ml.Animals were fixed with 10 mM sodium azide and examined by Nomarski or stereoscopic microscopy (Zeiss).Transgenic animals bearing the gfp or rfp reporter were observed by fluorescence microscopy (Leica MZFL III and Zeiss Axioplan2).Motility was evaluated by the waves of body bending per minute in M9 buffer using 15 young adult animals of two independent lines.Protein sample was prepared from 10 young-adult animals and used for western blot with anti-TNI-2 antiserum (clone R7; 50 ng/ml) as described (Ruksana et al., 2005).Density of the bands blotted was calculated by NIH Image software (http:// rsb.info.nih.gov/nih-image/).Data was presented as the means ± standard deviation and statistical analysis was performed with an unpaired Student's t-test.Difference at **P < 0.01 was considered to be statistically significant.

Sequence analysis
The cis-element search on the pro-Expression and mutation rescue of body-wall TNIs moter of three body-wall TNI genes in C. elegans and the search of the tni-3 head enhancer in C. elegans and sister species C. briggsae and C. remanei were performed using UCSC Genome Browser (http://genome.ucsc.edu/).Sequence alignments of DNA and amino acid were performed by ClustalW (http://www.genome.jp/tools/clustalw/). Amino acid sequences of C. elegans TNIs used: TNI-1; NM_077505, TNI-2; NM_077087, TNI-3; NM_074849, and TNI-4; NM_068340.

Expression patterns of three isoforms of body-wall TNI genes
We have reported that the tni-3 gene, that encodes one of the body-wall type TNIs, is strongly expressed in head muscles (Ruksana et al., 2005).In this study, we addressed the question how body-wall type TNI genes are expressed in the head region.Tissue expression patterns of three body-wall TNI genes were observed by using co-expressing green-and red-fluorescent reporter fusion genes (gfp and rfp).Reporter plasmids of pI1U2061R, pI2U2624 and pI3U3698R were constructed by fusing tni-1 with 2,061 bp upstream to rfp, tni-2 with 2,624 bp upstream to gfp and tni-3 with 3,698 bp upstream to rfp respectively (see MATERIALS AND METHODS).The pI2U2624 tni-2::gfp reporter and the pI3U3698R tni-3::rfp reporter were expressed in bodywall muscles (Fig. 1, A-C) including head region (Fig. 1, D-F), vulval (Fig. 1, G-I) and rectum muscles (Fig. 1, J-L), and the intense expression of the tni-3::rfp reporter was observed in the head region of body-wall muscles, vulva and rectum (Fig. 1, E, H and K, respectively).On the other hand, the expression of the pI1U2061R tni-1::rfp reporter was only observed in body-wall muscles as aggregated signals, compared with the tni-2::gfp expression (Fig. 1, M-O).These results suggest that TNI-1 functions only in body-wall muscle, but TNI-2 and TNI-3 function more widely in body-wall muscles including vulval and rectal muscles.

Promoter deletion analysis of the tni-3 gene and determination of the tissue-specific enhancer regions
As for the body-wall type TNI genes, the expression patterns and the tissue-specific enhancer regions of tni-1 and tni-2/unc-27 have been identified (Fig. 2A; Ruksana et al., 2005).To determine enhancer regions for the tni-3 expression at the sequence level, here we searched for cis-elements within the upstream regions of the three body-wall type TNI isoform genes using UCSC Genome Browser (http://genome.ucsc.edu/).pI3U2139R, which contains the upstream from -2,139 bp to the second exon of the tni-3 gene with the rfp reporter gene was expressed in vulval muscles, but pI3U1269R having much shorter upstream sequences was weakly expressed.Also, pI3U1269R was expressed in head muscles, but pI3U1100R was weakly expressed (Fig. 2A).
Summarized together, we found that vulva and head enhancer regions locate in the 871-bp-length from -1,269 bp to -2,139 bp and in the 169-bp-length from -1,100 bp to -1,269 bp, respectively.The vulva enhancer region includes three E-boxes (CANNTG), a 1275-enhancer element that is an HLH-1 target site, and an NdE-box (CATATG) that is the target site of HLH-8 (Fig. 2A; Krause et al., 1994;Harfe and Fire, 1998).The head enhancer region includes also an E-box and two-tandem copies of the CBF1/Su(H)/LAG-1-binding motif ((A/ G)TGGGAA) (Fig. 2, A and B; Christensen et al., 1996).Corresponding sequences in the head enhancer region in Fig. 2. Expression patterns of the promoter deletion constructs of three isoforms of body-wall type troponin I gene and identification of head and vulva enhancers on the upstream region of tni-3.pI3U3698R, pI3U2139R, pI3U1269R, pI3U1100R and pI3U769R as tni-3::rfp gene with respective promoter were expressed in the animal (Fig. 1).(A) Right column besides the scheme of promoter deletion constructs with cis-elements shows the numbers of cis-elements and expression profile of the three isoforms of body-wall type troponin I gene as the signal intensity of β-galactosidase (lacZ) and green and red fluorescent proteins (gfp and rfp) indicated by the number of symbols "+" or "-".VE, vulva enhancer; HE, head enhancer; BW, body-wall.Data for tni-1 and tni-2 was modified from Ruksana et al. (2005).pI1U2061R as tni-1::rfp gene with 2.0-kb-promoter was expressed in animal.pI2U2624 as tni-2::gfp with 2.6-kb-promoter and pI2U653 as tni-2::lacZ with 0.6-kb-promoter were expressed in animals.E, E-box; NdE, NdE-box; 1275 and 1330, the enhancer sequences of hlh-1 (Krause et al., 1994) 2B).We also searched within the upstream region of the human troponin I isoform-3 (TNNI3) gene.Three E-boxes and two CBF1/Su(H)/LAG-1-binding motifs are located at the upstream positions; CAGCTG at -107 bp and -763 bp, CAAGTG at -309 bp, and GAGGGAA at -408 bp, CTGG-GAA at -799 bp, respectively.These results indicate that E-box and CBF1/Su(H)/LAG-1-binding motifs are conserved from the worm to human, suggesting that the combination of both elements could be involved in the tni-3 expression in head region of animals.

C omparison of expression pattern and th e upstream transcription-factor binding sites in three TNI genes
In our previous report the expression patterns of four TNI genes and their regulatory sequences were analyzed (Ruksana et al., 2005).We summarize the locations of enhancer-or transcription-factor binding sites of the three body-wall type TNI genes and their expression profiles in Fig. 2A.The data clearly show that the expression differences between tni-1 and tni-2 are derived from the number of E-box, 1275 and 1330 sequences, which are known CeMyoD/HLH-1 binding sites.The absence of tni-1 in the vulva expression is consistent with a lack of the vulval enhancer NdE-box sequence in the promoter.Comparison between tni-2 and tni-3 clearly show that the head enhancer CBF1/ Su(H)/LAG-1-binding sequence occurs in tni-3 but not tni-2.These differences come from the gene duplication pro-  Yeast one-hybrid screening for isolating the head enhancer-sequence binding protein To isolate potential proteins regulating head muscle expression, the 169bp-fragment of the head enhancer-sequence from -1,100 bp to -1,269 bp upstream of tni-3 was employed as a bait for yeast one-hybrid screening (see MATERIALS AND METHODS).Eight plasmids were identified by screening approximately 2.0 × 10 5 colonies of yeast transformants (Table 1).Of these, four clones of lag-1 (Lin-12 and Glp-1 phenotype 1) were isolated as the head enhancer-sequence binding protein.The remaining four plasmids contained cDNAs from potential transcriptional regulators including max-2/Pak-1 (p21-activated kinase 1), stip-1 (septin and tuftelin-interacting protein 1), larp-5 (La ribonucleoprotein domain family, member 5) and M03C11.8(Table 1).These results suggest that lag-1 could bind an enhancer of tni-3 and may have an important role in the head expression of tni-3, although further experiments are required to confirm this.

Assessments of the functional complementation among three body-wall TNIs by recovery of motility in unc-27(e155) null mutant worms
The TNI-1, TNI-2 and TNI-3 isoforms are classified as body-wall muscle type by their expression pattern, amino acid sequence homology and cross-reactivity to other troponin components (Ruksana et al., 2005;Kagawa et al., 2007;Amin et al., 2007).However, their expression patterns are fairly different as was observed above (Fig. 2).To clarify whether or not TNI isoforms are interchangeable we tested their functional complementation (see MATE-RIALS AND METHODS).Each of the four TNI isoforms was expressed under the control of the tni-2/unc-27 promoter in the locomotion-defective unc-27(e155) null mutant animals (Fig. 3).Relative protein expression levels of TNI isoforms driven by tni-2/unc-27 promoter were determined by the signal intensity in the Western blots using anti-TNI-2 antibody, which cross-reacts with TNI-1, TNI-2 and TNI-3 (Ruksana et al., 2005;Amin et al., 2007).Quantities of TNI-1, TNI-2 and TNI-3 in transformed unc-27(e155) null animals were 42%, 106% and 49% compared TNI expression in wild-type N2 animal set at 100% (Fig. 3A).These data suggest that TNI-2 protein functioned more efficiently in transformed animals.
The binding sites of TNIs for physiological interacting to the other troponins were analyzed based on the amino acid sequence level (Fig. 4; Ruksana et al., 2005;Amin et al., 2007).The TNC-and Actin/TNC-binding sites of four TNIs were highly conserved at > 71% and > 94% homologies, respectively.On the other hand, the TNTbinding sites of TNI-1 and TNI-2 were highly conserved at 72% homology, whereas these of TNI-1 and TNI-3 or TNI-1 and TNI-4 were weakly conserved at 59% or 48% homologies, respectively.Furthermore, although the Cterminal extension with glutamate-repeats (E 4-11 ) was specific in three body-wall TNIs, the C-terminal extensions of TNI-1 and TNI-3 were weakly conserved at 43% homology.Therefore, in body-wall muscle, the TNI-TNT bindings and unknown functions of the C-terminal extension of TNIs would be strongly related to the motility of animals.

DISCUSSION
In this study, we addressed gene expression pattern, cis-and trans-regulatory elements and functional complementation for three body-wall muscle type TNIs.This revealed that TNI-3 was strongly expressed in head bodywall muscles and had an intermediated protein function among the three isoforms.

Tissue-specific enhancer for the expression of tni-3
in the head region and vulva Tissue-specific expression of tni-3 in the head region was regulated by the head enhancer which contains an E-box and a CBF1/Su(H)/ LAG-1-binding motif and expression in the vulva by the enhancer that contains an NdE-box and the 1275enhancer element (Fig. 2).HLH-1 binding to the E-box functions as a general enhancer for genes in all striated muscles including body-wall, vulva and rectum muscles (Krause et al., 1994), whereas HLH-8 binding to the NdEbox functions in a tissue-specific manner in vulval muscle formation (Kostas and Fire, 2002).The observation that one gene encodes one product in each of the four TNI genes is well known for many other muscle genes (Ruksana et al., 2005;Kagawa et al., 2007).This is the reason why each of the genes has different upstream con-trol sequences.New-gene finding studies allow sequence comparisons and phylogenic trees of coding regions.Our results strongly suggest that gene duplication processes should be studied as part of the evolution of promoter control mechanisms in multicellular eukaryote.
Interestingly, lag-1 encodes a homolog of the Drosophila Suppressor of Hairless (Su(H)) transcription factor in Notch signaling (Christensen et al., 1996).As described above the head enhancer-sequence contains a potential CBF1/Su(H)/LAG-1-binding motif, suggesting that LAG-1 may regulate expression of head muscle genes.LAG-1 is known to regulate the expression of the T-box transcription factor tbx-2 since gfp expression driven by a 5.2-kbpromoter containing the CBF1/Su(H)/LAG-1-binding motif was observed in the head regions of body-wall and pharynx (Smith and Mango, 2007).LAG-1 also activates and represses hlh-6 expression in pharyngeal gland and non-gland cells, respectively, under Notch signaling in a tissue-specific manner in the head of C. elegans (Ghai and Gaudet, 2008).Taken together, these suggest that the tni-3 expression is regulated by LAG-1 through CBF1/ Su(H)/LAG-1-binding motif in the head enhancer and TNI-3, in addition to motility, might also control head movement, which awaits future studies.

Relations between cell fate and genetic lineage:
Correlation between muscle development and cancer proliferation programs Interestingly, four of five candidates identified from the yeast one-hybrid screening for a head-enhancer binding protein of tni-3 were related to human cancers including hematological malignancy and solid carcinoma.CBF1/Su(H)/LAG-1 and LARP5 are highly expressed in human acute lymphoblastic leukemia (ALL) (Joshi et al., 2009;Bousquet-Antonelli and Deragon, 2009).Also, CBF1 is expressed in colon cancer cells and silencing of Notch/CBF1 signaling by γ-secretase inhibitors enhances taxane-induced mitotic arrest and apoptosis (Akiyoshi et al., 2008).In C. elegans, LET-60/ Ras, an oncogene in at least 70% of human colon cancers, induces RGL-1/RAL-1 activity and RAL-1 cooperates with Notch to determine vulva cell fate (Zand et al., 2011).AF-4/MAX-2 and STIP-1 are highly expressed in mixedlineage leukemia (MLL) and ovarian cancer cells, respectively (Chen et al., 1993;Kim et al., 2010).These results suggest that muscle development and cancer proliferation are correlated with each other by the mechanisms of stimulus-dependent regeneration.Elucidations of these molecular functions and a relationship between muscle and cancer developments would help understand how genes are evolved to control cell fate and cell growth.
We have previously reported that expression of the tropomyosin gene tmy-1/lev-11 in the pharynx and intestine is regulated by a combination of the ELT-2, CdxA and PHA-4 transcription factors.The binding sites of these transcription-factors are located in the internal promoter regions of the gene.Tissue-specific transcription can be stage-and tissue-specifically controlled by the existence or absence of candidate components in cells.Even the upstream region of a single gene contains many transcription factor-binding sites (Anokye-Danso et al., 2008).Taken together these results suggest that complex formation of the general muscle enhancer HLH-1 and the tissue-specific regulators HLH-8 or LAG-1 could control the tissue-specific expression of tni-3.
Functional complementation and sequence differences among three body-wall TNIs: An evolution model of four TNI genes Mutation rescue experiments using the different isoforms of the three body-wall type TNI genes indicate that the biological function and sequence homology of TNI-3 is different from the other body-wall type isoforms (Fig. 3).TNI-1 and TNI-2 have a similar function for body-wall muscle contraction, whereas TNI-3 has a weaker function than those of TNI-1 and TNI-2 as expected from their amino acid sequence homology (Ruksana et al., 2005).Since tni-3 is highly expressed in the body-wall muscles of the head region, TNI-3 may also function for the periodic movement of head, which awaits future studies.Pharyngeal muscle type tni-4 did not rescue motility because TNI-4 does not interact with other body-wall muscle components including TNC-1, TNT-1 and TMI (Ruksana et al., 2005;Amin et al., 2007).The in vivo rescue experiments (Fig. 3) confirm the results from the previous in vitro molecular interaction analysis (Ruksana et al., 2005;Amin et al., 2007).Briefly, TNI-3 and TNI-4 did not rescue animal motility, indicating that TNI-3 is a primitive body-wall type derived from the pharyngeal muscle type TNI-4.TNI-1 and TNI-2 rescued the motility, indicating that these two TNI isoforms are definitive body-wall types derived from TNI-3, and finally, TNI-2 was defined as the major type for expression and function in the body-wall muscles.Key point of our conclusion comes from combined results between molecular sequence analysis and developmental expression profiles.Early expressing gene, TNI-4 in pharynx is a primitive type of isoform and late expressing genes, three TNIs in body-wall appeared as more specialized isoforms.TNI-1 and TNI-2 close to each other.In this study we characterize differences between major body-wall type TNI-2 and an intermediate type TNI-3.This could be related to "an individual development repeats the phylogenic developments".The molecular function of TNI-3 could be altered in a tissuespecific manner according to changes in tni-3 expression that could be caused by additional head enhancer and/or vulva enhancer elements in the upstream sequences.Our results confirm that coding sequences and the upstream sequences of enhance are also important for expressions and functions of the gene.TNI-2, in addition to TNI-1, has a major role in body-wall muscle con-traction, so explain why the region-specific TNI-3 isoform did not rescue the unc-27 null mutant phenotype.
The experimental approach of exchanging one gene family member for another isoform or splicing variant with tissue-specific promoters could be a powerful tool for understanding tissue specificity of expression and molecular interactions, and this is the first study for analyzing molecular and biological functions of coding and noncoding regions of three homologous genes, such as gene expression pattern and animal behavior.Furthermore, interspecies complementation studies such as functional molecular evolution of coding and noncoding regions may be a strategy that would provide useful information regarding gene and/or genome evolution among species.
Fig.2.Expression patterns of the promoter deletion constructs of three isoforms of body-wall type troponin I gene and identification of head and vulva enhancers on the upstream region of tni-3.pI3U3698R, pI3U2139R, pI3U1269R, pI3U1100R and pI3U769R as tni-3::rfp gene with respective promoter were expressed in the animal (Fig.1).(A) Right column besides the scheme of promoter deletion constructs with cis-elements shows the numbers of cis-elements and expression profile of the three isoforms of body-wall type troponin I gene as the signal intensity of β-galactosidase (lacZ) and green and red fluorescent proteins (gfp and rfp) indicated by the number of symbols "+" or "-".VE, vulva enhancer; HE, head enhancer; BW, body-wall.Data for tni-1 and tni-2 was modified fromRuksana et al. (2005).pI1U2061R as tni-1::rfp gene with 2.0-kb-promoter was expressed in animal.pI2U2624 as tni-2::gfp with 2.6-kb-promoter and pI2U653 as tni-2::lacZ with 0.6-kb-promoter were expressed in animals.E, E-box; NdE, NdE-box; 1275 and 1330, the enhancer sequences of hlh-1(Krause et al., 1994); LAG-1, CBF1/Su(H)/LAG-1-binding motif.(B) Sequence alignment of the head enhancer region in C. elegans and corresponding sequences in C. briggsae and C. remanei.Asterisk (*) indicates identical nucleotide.Gray box, E-box (CANNTG); under bar, CBF1/Su(H)/LAG-1-binding motif ((A/G)TGGGAA).

Fig. 4 .
Fig. 4. Sequence alignment and structural homology of the C. elegans body-wall and pharynx TNIs.(A) Sequence alignment of TNIs of C. elegans.The shade indicates the TNC-, TNT-and Actin/TNC-binding sites and the body-wall specific C-terminal extension with glutamate-repeats, respectively.The identity and similarity of the amino acid residues are indicated by (*) and (:/.), respectively.(B) Structural homology of the binding sites of TNIs.Table represents percentage homology of each binding sites of four TNIs, compared with TNI-1.BW; body-wall, PHX; pharynx.

Table 1 .
Summary of yeast one-hybrid screening for the head-specific expression of tni-3