The Erk MAP kinase pathway is activated at muscle spindles and is required for induction of the muscle spindle-specific gene Egr3 by neuregulin1.

Muscle spindles are sensory receptors composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of sensory neuron axons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. The transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming intrafusal fibers. We have previously shown that, in cultured muscle cells, NRG1 signaling activates the Egr3 gene through SRF and CREB, which bind to a composite regulatory element, and that NRG1 signaling targets SRF by stimulating nuclear translocation of SRF coactivators myocardin-related transcription factor (MRTF)-A and MRTF-B and targets CREB by phosphorylation. The current studies examined signaling relays that might function in the NRG1 pathway upstream of SRF and CREB. We found that transcriptional induction of Egr3 in response to NRG1 requires the MAP kinase Erk1/2, which acts upstream of CREB to induce its phosphorylation. MRTFs are targeted by the Rho-actin pathway, yet in the absence of Rho-actin signaling, even though MRTFs fail to be translocated to the nucleus, NRG1 induces Egr3 transcription. In mouse muscle in vivo, activation of Erk1/2 is enhanced selectively where muscle spindles are located. These results suggest that Erk1/2 acts in intrafusal fibers of muscle spindles to induce transcription of Egr3 and that Egr3 induction occurs independently of MRTFs and involves Erk1/2 acting on other transcriptional regulatory targets that interact with the SRF-CREB regulatory element.

Neuregulin1 signaling targets SRF and CREB and activates the muscle spindle-specific gene Egr3 through a composite SRF-CREB-binding site.

Muscle spindles are sensory receptors embedded within muscle that detect changes in muscle length. Each spindle is composed of specialized muscle fibers, known as intrafusal muscle fibers, along with the endings of axons from sensory neurons that innervate these muscle fibers. Formation of muscle spindles requires neuregulin1 (NRG1), which is released by sensory axons, activating ErbB receptors in muscle cells that are contacted. In muscle cells, the transcription factor Egr3 is transcriptionally induced by NRG1, which in turn activates various target genes involved in forming the intrafusal fibers of muscle spindles. The signaling relay within the NRG1-ErbB pathway that acts to induce Egr3 is presumably critical for muscle spindle formation but for the most part has not been determined. In the current studies, we examined, using cultured muscle cells, transcriptional regulatory mechanisms by which Egr3 responds to NRG1. We identified a composite regulatory element for the Egr3 gene, consisting adjacent sites that bind cAMP response element binding protein (CREB) and serum response factor (SRF), with a role in NRG1 responsiveness. The SRF element also influences Egr3 basal expression in unstimulated myotubes, and in the absence of the SRF element, the CREB element influences basal expression. We show that NRG1 signaling, to target SRF, acts on the SRF coactivators myocardian-related transcription factor (MRTF)-A and MRTF-B, which are known to activate SRF-mediated transcription, by stimulating their translocation from the cytoplasm to the nucleus. CREB is phosphorylated, which is known to contribute to its activation, in response to NRG1. These results suggest that NRG1 induces expression of the muscle spindle-specific gene Egr3 by stimulating the transcriptional activity of CREB and SRF.

Chromatin modifications that support acetylcholine receptor gene activation are established during muscle cell determination and differentiation.

Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms. An important way of regulating transcription is through targeting histone modifications on chromatin to distinct gene loci. Using chromatin immunoprecipitation, we examined the developmental regulation of certain histone modifications at the AChR epsilon subunit locus, including methylations at lysine residues K4 and K27 and acetylations at K9 and K14. We modeled various stages of muscle development in cell culture, including pre-determined cells, committed but undifferentiated myoblasts, and differentiated myotubes, and modeled synaptic myotube nuclei by stimulating myotubes with neuregulin (NRG) 1. We found that a pattern of histone modifications associated with transcriptional activation is targeted to the AChR epsilon subunit locus in myotubes prior to stimulation with NRG1 and does not change upon addition of NRG1. Instead, we found that during muscle cell determination and differentiation, specific histone modifications are targeted to the AChR epsilon subunit locus. Within the gene, at K4, dimethylation is induced during muscle cell determination, while trimethylation is induced during differentiation. At K27, loss of trimethylation and appearance of monomethylation occurs during determination and differentiation. In addition, in a region upstream of the gene, K4 di- and trimethylation, and K9/14 acetylation are induced in a distinct developmental pattern, which may reflect a functional regulatory element. These results suggest synaptic signaling does not directly target histone modifications but rather the histone modification pattern necessary for transcriptional activation is previously established in a series of steps during muscle development.

Neuregulin-1 induces acetylcholine receptor transcription in the absence of GABPalpha phosphorylation.

Localization of acetylcholine receptors (AChRs) to the postsynaptic region of muscle is mediated in part by transcriptional mechanisms, because the genes encoding AChR subunits are transcribed selectively in synaptic myofiber nuclei. Neuregulin-1 (NRG-1) is a synaptic signal and induces transcription of AChRs in muscle cells. Signaling by NRG-1 is thought to involve the transcription factor GA-binding protein (GABP), a heterodimer of GABPalpha, which is a member of the Ets family, and GABPbeta. Phosphorylation of certain other Ets proteins outside the Ets DNA-binding domain serves to stimulate transcriptional activation in response to extracellular signals. According to previous studies, NRG-1 stimulates phosphorylation of GABPalpha at threonine 280 in the N-terminal region adjacent to the Ets domain, suggesting that GABPalpha phosphorylation might contribute to NRG-1 responsiveness. To determine the functional importance of the N-terminal region of GABPalpha and whether its function is regulated by phosphorylation, we generated muscle cell lines in which the endogenous GABPalpha gene was deleted and replaced by variants of GABPalpha mutated in the N-terminal region. We found that NRG-1 can induce transcription in cells with mutated T280 phosphorylation site, indicating that T280 phosphorylation does not contribute to NRG-1 responsiveness. We also found that NRG-1-induced transcription occurs in cells missing the entire N-terminal region of GABPalpha. Because NRG-1 signaling is not expected to alter the function of the C-terminal region, which remains in these cells, these results suggest that GABPbeta, or other interacting components, rather than GABPalpha directly, is targeted by NRG-1 signaling.

Directing RNA interference specifically to differentiated muscle cells.

A common approach for mediating RNA interference (RNAi) is to introduce DNA that encodes short hairpin RNA (shRNA), which is often contained in a plasmid that can express a shRNA in a wide variety of cell types. Muscle cells and certain other cell types grown in culture can exist in both a dividing state and in a post-mitotic, differentiated state, and it is sometimes useful to induce RNAi selectively in terminally differentiated cells to study the function of a gene, particularly when the gene is also required for propagation of dividing cells. We describe two methods for studying gene function by RNAi specifically in terminally differentiated skeletal muscle cells in culture. We developed a shRNA expression vector, based on myosin light chain 1f gene regulatory sequences, which is designed to induce shRNA expression specifically after differentiation has been initiated. We show that this vector can mediate RNAi and is only active in differentiated muscle cells. Also, we developed an adenoviral vector that is designed to be able to deliver shRNAs directly to post-mitotic muscle cells. We show that adenoviruses produced using this vector mediate RNAi in differentiated muscle cells. These methods add to the repertoire of RNAi tools that can be used for identifying genes involved in any event of interest that occurs in differentiated muscle cells.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Neuregulin-1 induces expression of Egr-1 and activates acetylcholine receptor transcription through an Egr-1-binding site.

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) and its receptors, ErbBs, are concentrated at synaptic sites, and NRG-1 activates AChR synthesis in cultured muscle cells, suggesting that NRG-1-ErbB signaling functions to activate synapse-specific transcription. Previous studies have demonstrated that NRG-1-induced transcription is conferred by cis-acting elements located within 100 bp of 5' flanking DNA from the AChR epsilon subunit gene, and that it requires a GABP binding site within this region. To determine whether additional regulatory elements have a role in NRG-1 responsiveness, we used transcriptional reporter assays in a muscle cell line, and we identified an element that is required for NRG-1-induced transcription (neuregulin response element, NRE). Proteins from myotube extracts bind the NRE and NRG-1 treatment of the cells stimulates this binding. The ability of NRG-1 to stimulate formation of a protein-DNA complex with the NRE requires induction of protein expression. The complex contains early growth response-1 (Egr-1), a member of the Egr family of transcription factors, because proteins in the complex bind specifically to an Egr consensus site, and formation of the complex is inhibited by antibodies to Egr-1. NRG-1 induces expression of Egr-1 in myotubes, which presumably is responsible for the ability of NRG-1 to stimulate protein binding to the NRE. These results suggest that NRG-1 signaling in myotubes involves induction of Egr-1 expression, which in turn serves to activate transcription of the AChR epsilon subunit gene.

Accelerated response of the myogenin gene to denervation in mutant mice lacking phosphorylation of myogenin at threonine 87.

Gene expression in skeletal muscle is regulated by a family of myogenic basic helix-loop-helix (bHLH) proteins. The binding of these bHLH proteins, notably MyoD and myogenin, to E-boxes in their own regulatory regions is blocked by protein kinase C (PKC)-mediated phosphorylation of a single threonine residue in their basic region. Because electrical stimulation increases PKC activity in skeletal muscle, these data have led to an attractive model suggesting that electrical activity suppresses gene expression by stimulating phosphorylation of this critical threonine residue in myogenic bHLH proteins. We show that electrical activity stimulates phosphorylation of myogenin at threonine 87 (T87) in vivo and that calmodulin-dependent kinase II (CaMKII), as well as PKC, catalyzes this reaction in vitro. We find that phosphorylation of myogenin at T87 is dispensable for skeletal muscle development. We show, however, that the decrease in myogenin (myg) expression following innervation is delayed and that the increase in expression following denervation is accelerated in mutant mice lacking phosphorylation of myogenin at T87. These data indicate that two distinct innervation-dependent mechanisms restrain myogenin activity: an inactivation mechanism mediated by phosphorylation of myogenin at T87, and a second, novel regulatory mechanism that regulates myg gene activity independently of T87 phosphorylation.

Inhibition of lens fiber cell morphogenesis by expression of a mutant SV40 large T antigen that binds CREB-binding protein/p300 but not pRb.

Simian virus (SV) 40 large T antigen can both induce tumors and inhibit cellular differentiation. It is not clear whether these cellular changes are synonymous, sequential, or distinct responses to the protein. T antigen is known to bind to p53, to the retinoblastoma (Rb) family of tumor suppressor proteins, and to other cellular proteins such as p300 family members. To test whether SV40 large T antigen inhibits cellular differentiation in vivo in the absence of cell cycle induction, we generated transgenic mice that express in the lens a mutant version of the early region of SV40. This mutant, which we term E107KDelta, has a deletion that eliminates synthesis of small t antigen and a point mutation (E107K) that results in loss of the ability to bind to Rb family members. At embryonic day 15.5 (E15.5), the transgenic lenses show dramatic defects in lens fiber cell differentiation. The fiber cells become post-mitotic, but do not elongate properly. The cells show a dramatic reduction in expression of their beta- and gamma-crystallins. Because CBP and p300 are co-activators for crystallin gene expression, we assayed for interactions between E107KDelta and CBP/p300. Our studies demonstrate that cellular differentiation can be inhibited by SV40 large T antigen in the absence of pRb inactivation, and that interaction of large T antigen with CBP/p300 may be enhanced by a mutation that eliminates the binding to pRb.

Inhibition of crystallin expression and induction of apoptosis by lens-specific E1A expression in transgenic mice.

Previous studies have shown that the adenovirus E1A oncoprotein can bind to and inactivate the retinoblastoma tumor suppressor protein (pRb) and the transcriptional coactivators CBP/p300. In this study, wild-type E1A12S or two deletion mutants (delN, which binds pRb but not CBP/p300; delCR2, which binds to CBP/p300 but not pRb) were linked to the lens-specific alphaA-crystallin promoter, and used to generate transgenic mice. Lens fiber cells expressing E1A12S or delCR2, both of which bind to CBP/p300, failed to upregulate beta-crystallin and gamma-crystallin expression. In contrast, lens fiber cells expressing delN showed significant expression of beta- and gamma-crystallins. Lens fiber cells expressing delN showed cell cycle entry, marked apoptosis, and evidence for p53 activation, while cells expressing either 12S or delCR2 showed limited apoptosis and no evidence for upregulation of the p53-inducible gene p21. Our results suggest that the transcriptional coactivators CBP and/or p300 are required for the dramatic increases in crystallin expression that accompany terminal differentiation in the lens, and also for activation of p53 in response to inactivation of pRb in the lens.