TnIfast IRE enhancer: Multistep developmental regulation during skeletal muscle fiber type differentiation

Abstract

To identify developmental steps leading to adult skeletal muscle fiber-type-specific gene expression, we carried out transgenic mouse studies of the IRE enhancer of the quail TnIfast gene. Histochemical analysis of IRE/herpesvirus tk promoter/␤-galactosidase reporter transgene expression in adult muscle directly demonstrated IRE-driven fast vs. slow fiber-type specificity, and IIB>IIX>IIA differential expression among the fast fiber types: patterns similar to those of native-promoter TnIfast constructs. These tissue-and cell-type specificities are autonomous to the IRE and do not depend on interactions with a muscle gene promoter. Developmental studies showed that the adult pattern of IRE-driven transgene expression emerges in three steps: (1) activation during the formation of primary embryonic (presumptive slow) muscle fibers; (2) activation, to markedly higher levels, during formation of secondary (presumptive fast) fibers, and (3) differential augmentation of expression during early postnatal maturation of the IIB, IIX, IIA fast fiber types. These results provide insight into the roles of gene activation and gene repression mechanisms in fiber-type specificity and can account for apparently disparate results obtained in previous studies of TnI isoform expression in development. Each of the three IRE-driven developmental steps is spatiotemporally associated with a different major regulatory event at the fast myosin heavy chain gene cluster, suggesting that diverse muscle gene families respond to common, or tightly integrated, regulatory signals during multiple steps of muscle fiber differentiation.

Activity-dependent repression of muscle genes by NFAT

“…This 130-bp chimeric enhancer was linked to the luciferase reporter and used to generate transgenic mice. As observed with the endogenous TnIf gene (15,20), the luciferase reporter was expressed in both the slow soleus and fast extensor digitorum longus (EDL) muscles during the first postnatal week (P7), and as the muscles matured, reporter activity became progressively more restricted to the EDL (Fig. 1B).…”

“…Like other genes encoding contractile proteins, TnI transcription during embryonic/ early fetal development is regulated by the MRF (i.e., MyoD) and MEF-2 families of factors that bind cis elements harbored in the SURE and FIRE enhancers (21,26,27). The differential expression of TnIs and TnIf in diverse fibers is already evident during late fetal and perinatal development (15,20), but their expression becomes more confined to slow-and fast-twitch myofibers after P7 when motoneurons depolarize muscles with distinct activity patterns (Fig. 1B and refs.…”

“…To this end, we chose to study the troponin I slow (TnIs) and troponin I fast (TnIf) genes because their expression is restricted to slow-or fast-twitch myofibers in adult muscle. TnIs and TnIf mRNAs are initially coexpressed in all muscle groups during embryonic development (15,20) and in differentiating cultured myocytes (21,22). Later, as developing myofibers are innervated and depolarized by motoneurons, their …”

“…A systematic deletion analysis in transgenic mice led to the identification of a 128-bp slow-specific upstream regulatory element (SURE) and a 144-bp fast-specific intronic regulatory element (FIRE) that restrict TnI transcription to either slow or fast muscles (20,26). Interestingly, these enhancers share four homologous cis-acting regulatory motifs: an E-box and MEF-2, CACC and CAGG sites (26,27).…”

“…The TnIs SURE is bipartite, with the half that contains the first three elements conferring general muscle specificity, whereas the other half harboring the CAGG site and a bicoid-like motif, which binds GTF-3, is required for confining transcription to slow muscle (15,27). Although the TnIf FIRE is sufficient to confer developmental and fiber-type-specific transcription (20,26), the cis elements conferring this specificity are not known.…”