Phylogenetic implications of the superfast myosin in extraocular muscles

Extraocular muscles (EOMs) are of special interest because they differ from other skeletal muscles in ontogeny, innervation and contractile physiology. For these reasons, they are likely to provide novel insights into the origin of specialized vertebrate muscles and into the evolution of striated muscle itself.

EOMs arise from unsegmented head mesenchyme rather than somites, which give rise to trunk and limb muscles (Noden et al., 1999). They are innervated by cranial, rather than spinal,motoneurons and are organized into motor units composed of fibers with different molecular and ultrastructural properties(Goldberg and Shall, 1999). Ultrastructural analyses have identified six fiber types in extraocular muscle(Peachey, 1971;Spencer and Porter, 1988), and the majority of EOM fibers are singly-innervated and phasic, like limb and trunk skeletal muscles, while a minority are multiply-innervated and tonic. But, irrespective of their innervation, almost all are heterogeneous in myosin expression (Jacoby et al.,1990; McLoon et al.,1999; Rubinstein and Hoh,2000) and none corresponds to the Type I and II fibers found in other skeletal muscles. Most importantly, they exhibit distinctive physiological properties, in particular, superfast contractions(Close and Luff, 1974;Li et al., 2000) that are linked to the expression of a novel superfast myosin heavy chain.

In many ways, the superfast EOM myosin is a molecular reflection of EOM's distinctive ontogenic, structural and physiological features. First, its expression is tissue-restricted: aside from extraocular muscle, it is found only in laryngeal muscles (Briggs and Schachat, 2000; Lucas et al.,1995). Second, in many EOM fibers, it is co-expressed with other myosins (Briggs and Schachat,2001; Rubinstein and Hoh,2000). And third, its localization to the central end-plate band region where EOM fibers are innervated links its expression directly to innervation (Briggs and Schachat,2001; Rubinstein and Hoh,2000). Here, the MYH13 gene that encodes this distinctive molecular marker is analyzed by genomic mapping, sequence-based phylogenetic techniques and phylogenetic footprinting to gain insights into the evolution of diversity and specialization in striated muscle myosins and the factors that probably account for the tissue-restricted expression of MYH13.

Cloning of the 3′-untranslated region of the extraocular (EO) myosin heavy chain mRNA from rabbit and rat EOM enabled us to design primers to amplify the 3′ untranslated region (3′-UTR) of the human and mouse EO MYH genes and localize them to human chromosome 17 on the Stanford G3 radiation hybrid panel and mouse chromosome 11 on the EUCIB (European Backcross) panel (Winters et al.,1998). Neighboring sequence-tagged sites more specifically localized the human EO MYH gene to 17p13.1 to p12, placing it within 4 centimorgans of the fast/developmental MYH gene cluster identified by Yoon et al. (1992), and further screening showed that the EO MYH gene resides on YAC B120C11, which contains other members of the fast/developmental cluster. These chromosomal analyses led to the designation of the human and mouse EO MYH genes as MYH13 and myh13,respectively (Winters et al.,1998).

The sequence of a full-length cDNA for the rabbit EOM MYH enabled us to identify the superfast myosin gene MYH from the human genome project sequences and to define its exon organization using GeneQuest (Briggs and Schachat,1999,2000;Schachat and Briggs, 1999). The exon boundaries of the other members of the fast/developmental MYH cluster— the embryonic, IIA, IIX, IIB and perinatal MYH genes — were similarly identified (Schachat and Briggs,1999). The resulting map of the fast/developmental MYH cluster is presented in Fig. 1. These maps reveal that the MYH13 gene differs significantly from its neighbors in two ways. First, it spans almost 64 kb, more than twice the length of any other member of the cluster (Fig. 1A); and second, with the exception of two short 5′ regions,MYH13 lacks the internal similarities in exon organization evident in other members of the cluster (Fig. 1B).

The conservation of exon size and phase (reading frame) among members of the fast/developmental MYH cluster (Table 1) enables regions of homologous intron—exon spacing to be identified. In Fig. 1B, these regions are indicated by common color; only two common patterns in the 5′ region are present in MYH13. Because these common spatial patterns probably reflect lineage (e.g. gene duplications) and genetic exchanges between neighboring members of the cluster, both the size and the non-homologous intron—exon organization of MYH13 indicate that it has been largely `insulated' from events that have shaped the current organization of the other members of the fast/developmental MYH gene cluster.

Ten striated myosin heavy chain genes have been localized in the human genome: the six members of the fast/developmental MYH gene cluster (the embryonic, IIA, IIX, IIB, perinatal and extraocular genes)(Weiss et al., 1999a); two slow/cardiac MYH genes α and β on human chromosome 14(Mahdavi et al., 1984;Saez et al., 1987); the masticatory myosin (IIM), an orphan striated muscle MYH gene on chromosome 7(Hoh et al., 1999), which appears to be a pseudogene encoding the IIM myosin in humans(Schachat and Briggs, 1999),and a gene that probably encodes the slow-tonic MYH on chromosome 20(Berg et al., 2001).Fig. 2,Fig. 2,Fig. 2,Fig. 2 presents an amino acid sequence alignment of the striated MYHs using Clustal X(Thompson et al., 1997), andFig. 3 shows the phylogeny inferred by neighbor-joining analysis. MYH IIM, the most divergent MYH gene sequence, was used as an outlier and, as depicted in the figure, bootstrapping analysis indicates that the phylogenetic tree is well-supported. In this phylogeny, MYH13 occupies a critical position. It arose from the precursor of the fast/developmental MYH genes shortly after the fast/developmental and slow cardiac precursors diverged. Thus, MYH13 represents an ancient MYH lineage. It appears to be the first member of the fast/developmental cluster to diverge and specialize, having arisen even before the developmental MYH genes.

Support for that ancient lineage is suggested by phylogenetic analysis of the two functionally distinct domains of striated muscle myosin. Previous studies have found that the sequences of other striated muscle myosin head or motor domains and their coiled-coil rod or tail domains generate similar phylogenetic relationships — implying that the these functionally distinct domains have evolved at similar rates(Korn, 2000). However, as shown in Fig. 4, the motor and rod domains of MYH13 do not follow this relationship: the rod is more closely related to the slow/cardiac MYHs than to the fast/developmental genes, while the motor domain is more similar to the fast/developmental MYHs. Coupled with the sequence phylogeny, this finding implies that the MYH13 gene arose before the fast/developmental and cardiac MYH genes had significantly diverged,possibly at a time when the precursors of the slow/cardiac and fast/developmental gene clusters were linked.

The exon organization of the MYH13 gene provides independent support for its strategic position in the radiation of mammalian striated muscle MYH genes. Its exon boundaries are identical in position and phase (reading frame)with those previously described for the rat and human embryonic MYHs(Stedman et al., 1990;Strehler et al., 1986). However, MYH13 differs from the adult fast and perinatal MYH genes in its 3′ coding exons (Fig. 1B;Table 1). The 49 C-terminal amino acid residues of MYH13 are encoded by two exons, exons 40 and 41; but the homologous sequences of the adult fast and perinatal MYHs are encoded by a single exon, exon 40. This single-exon pattern is also shared with the slow/cardiac genes (Epp et al.,1993; Liew et al.,1990), providing independent support for the inference from sequence phylogeny that MYH13 and the slow/cardiac genes arose from duplications of a common ancestor — one that possessed a `primitive'3′-exon organization rather than the `derived' single-exon organization of the perinatal and adult fast MYHs(Schachat and Briggs,1999).

In addition to its 3′-exon organization and the `insulation'suggested by its exon spacing, MYH13 exhibits distinctive transcriptional features that distinguish it from the other members of the fast/developmental gene cluster and from the slow/cardiac MYH genes. Analysis of rabbit full-length MYH13 cDNAs (Briggs and Schachat, 2000) indicates that transcription can initiate at three sites (Fig. 5). Transcripts A and B generate 5′-UTRs that are significantly longer than the single 5′-UTRs reported for other MYH genes. Comparison of transcripts B and C with A shows they are shorter and each incorporates an additional 50-nucleotide insertion that corresponds to a 5′-non-coding exon present in the human EO myosin cDNA (Weiss et al.,1999b). Although alternative splicing of the 5′-UTR sequences and multiple transcription initiation sites have been described for several muscle genes including enolase, cardiac troponin T and acetylcholinesterase, where they are important for the developmental or regional regulation of gene expression(Giallongo et al., 1993;Jin et al., 1995;Li et al., 1993;Takechi et al., 1994), this is the only striated muscle MYH gene for which it has been described.

Genomic sequence analysis also provides insights into the mechanisms that may account for the tissue-restricted patterns of MYH13 expression. Promoter and enhancer analysis of the embryonic, cardiac and IIB MYH genes has identified transcription factors that regulate striated-muscle-specific expression. These include the myogenic factors MyoD, myogenin and myf-5, which bind E-box sequences, and binding sites for other key transcription factors such as MEF2, TEF, SP1 and Oct1 (Bouvagnet et al., 1987; Firulli and Olson, 1997; Gossett et al.,1989; Gupta et al.,1998; Shimizu et al.,1992; Swoap, 1998;Takeda et al., 1992). Generalizing from the organization of regulatory elements described for the IIB and cardiac MYH genes, as well as many other genes(Blackwood and Kadonaga, 1998),the binding sites for these factors generally cluster in a proximal promoter within several hundred nucleotides upstream of the transcription initiation site and distal regions that function as transcriptional enhancers or suppressors. These regions typically lie 1-2kb upstream of the promoter and in the first or second intron, although they may reside beyond the 3′ end of the gene, as is the case for myosin light chain 1f/3f(Donoghue et al., 1988). The initial inspection of these regions of the MYH13 gene in the human genome suggested that it was regulated differently(Briggs and Schachat, 2000). Strikingly, only a single binding site for these factors, an E-box, was present in the proximal promoter region(Fig. 6). This led us to propose that MYH13 expression might be directed by a novel set of transcription factors.

Because its expression is restricted to extraocular and laryngeal muscle,regulation of the EO MYH must differ from that of the other fast/developmental and slow/cardiac MYHs. Minimally, this means that factors other than the myogenic transcription factors must also be involved in determining when and where MYH13 is transcribed. The co-expression of MYH13 with adult fast myosins in extraocular muscle and laryngeal muscle fibers, coupled with its absence from limb fibers expressing the adult fast myosins, serves to emphasize its requirement for regulatory factors other than those that drive the trunk and limb muscle MYH genes.

Insights into the nature of that regulation come from the identification of conserved sequences in the 5′ flanking and intronic regions of the human MYH13 and mouse myh13 genes. This comparative genomic technique, known as phylogenetic footprinting, is based on the observation that important regulatory sequences are conserved across species and has been used to detect muscle-specific regulatory sequences(Wasserman and Fickett, 1998). Using BLAST 2 (Tatusova and Madden,1999), several highly conserved regions were identified, including a likely proximal promoter, which includes 250bp upstream of the transcription start sites, as well as several possible enhancer or repressor regions upstream and in the first two introns(Fig. 7).

The potential transcription factor binding sites in these regions were characterized with the matInspector 2.2(Quandt et al., 1995) and GeneQuest (DNAstar, Madison, WI, USA) programs, which compare a test sequence with a large database of consensus binding sites. Many potential sites are clustered in the conserved regions. Among the most frequent consensus sequences present in both the mouse and human genomic sequences are those for NF1, which directs aldolase expression in a subset of fast fibers(Salminen et al., 1996),Pitx2, which is necessary for EOM embryonic development(Kitamura et al., 1999),NKX2.5, which is a mammalian homolog of tinman, a regulator of cardiac development (Schwartz and Olson,1999), TCF11, which binds a DNase-hypersensitive site in theβ-globin cluster (Johnsen et al.,1998), and delta EF1, which inhibits myoD-promoted myogenesis(Sekido et al., 1994). Some myogenic factor binding sites are also present, but they do not generally occur in the clusters that allow for myogenic factor dimerization that is critical for activating other MYH genes. The Pitx2, NKX2.5, TCF11 and delta EF1 consensus binding sites are particularly interesting given the early specialization of the EO MYH13 gene, its phylogenetic relationship to the slow/cardiac MYH genes, whose transcription is not regulated by myogenic factors, and the myoD-independent development of EOM revealed in studies on the myoD/myf5 knockout mouse (Tajbakhsh et al., 1997). The presence of these conserved sites suggests that MYH13 expression may be integrated into a broader more primitive developmental program that directs the formation of EOMs or of the eyes themselves. This is consistent with the finding that NKX2.5, which regulates cardiac development,is expressed at high levels in extraocular muscle and the fact that mammalian homologs of factors that control eye development in Drosophila,including Dach2, Six1, Eya2 and Pax3, have been found to participate in a regulatory network that directs myogenic factor expression in vertebrate somites (Heanue et al.,1999).

In addition to promoter and enhancer elements, some genes are regulated by additional elements that control their chromatin conformation. An open chromatin conformation is a necessary prerequisite for transcription. Open conformations may be generated locally in the proximal promoter by the action of histone deacetylases associated with the transcription complex, but the chromatin conformation of some tissue-specific genes is controlled by distinct upstream and downstream elements that specify the conformation of entire chromosomal domains of 30-100 kb or more(Blackwood and Kadonaga, 1998). The boundaries of these chromodomains (as well as active proximal promoters)are characterized by DNase hypersensitivity and may also be marked by CpG islands (Blackwood and Kadonaga,1998; Lewin,1994). Phylogenetic footprinting of MYH13 gene structure reveals the presence of such chromodomain-delimiting CpG islands(Fig. 8). Interestingly, the upstream CpG island (11 kb upstream from the human proximal promoter) overlaps with the 3′ region of an 800-nucleotide non-coding sequence that exhibits greater than 75% sequence identity between human and mouse. This surprising homology, coupled with the apparent `insulation' of the extraocular myosin heavy chain gene from the other members of the fast/developmental MYH cluster, suggests that MYH13 may reside in a chromodomain that occurs in a constitutively closed conformation that represses its transcription in trunk and limb muscle. The analysis of the chicken β-globin genes provides precedence for such chromatin-dependent regulation of a single member of a multigene cluster (Felsenfeld,1993; Hebbes et al.,1994).

In Fig. 9, the phylogenetic relationships and gene order of the fast/developmental cluster are modeled as a series of spatially ordered gene duplications that can generate the fast/developmental MYH cluster (Fig. 1A). Beginning with the duplication of the precursor of the slow/cardiac and fast/developmental clusters, it proceeds to duplications that generate the extraocular MYH gene, then the developmental MYH genes and finally the adult fast MYH genes. This model for the origin of the fast/developmental MYHs differs from previous models for the origin of the fast/developmental cluster because it takes into account the critical position of MYH13 near the divergence of the slow/cardiac and fast/developmental MYH genes.

We wish to thank Mr Amar Nijagal for his assistance and the National Eye Institute (USA) for award EY11377 which funded this research.