In adult skeletal muscle, satellite cells reside beneath the basal lamina of muscle, closely juxtaposed to muscle fibers, and make up 2 to 7 percent of the nuclei associated with a particular fiber. Satellite cells are normally mitotically quiescent but are activated (they enter the cell cycle) in response to stress induced by weight bearing or by trauma. The descendants of activated satellite cells, called myogenic precursor cells, undergo multiple rounds of division before fusion and terminal differentiation. As defined by biological, biochemical, and genetic criteria, satellite cells are distinct from their daughter myogenic precursor cells. Activated satellite cells also generate progeny that restore the pool of quiescent satellite cells.
The maintenance of satellite cell numbers in aged muscle after repeated cycles of degeneration and regeneration has been interpreted to support the notion that satellite cells possess an intrinsic capacity for self-renewal. Asymmetric distribution of the Numb protein in daughters of satellite cells in cell culture has been implicated in the asymmetric generation of distinct daughter cells for self-renewal or differentiation. However, whether satellite cells are true stem cells or, alternatively, dedifferentiated myoblasts remains unresolved.
Our laboratory discovered that satellite cells express the transcription factor Pax7. Members of the paired-box family of transcription factors (Pax1-9) have important functions in regulating the development and differentiation of diverse cell lineages during embryogenesis. Pax7 and the closely related Pax3 gene are paralogues, encoding proteins with almost identical amino acid sequences and partially overlapping expression patterns during mouse embryogenesis. Notably, Pax3 plays an essential role in regulating the developmental program of MyoD-dependent migratory myoblasts during embryogenesis. More recently, it has been suggested that Pax3+/Pax7+ progenitors originating in the embryonic somite are the precursors of satellite cells in adult muscle.
Pax7 and Pax3 proteins bind to similar, if not identical, sequence-specific DNA elements, suggesting that they regulate similar sets of target genes. Furthermore, gain-of-function mutations in and increased expression of Pax3 and Pax7 are associated with the development of alveolar rhabdomyosarcomas, indicating that both molecules regulate similar activities in the myogenic program. Although the Pax3 and Pax7 proteins are structurally similar, analysis of null mutations in mice indicates that they are required for the development of a number of distinct cell lineages and appear to have nonredundant roles in myogenesis.
We performed an extensive analysis of Pax7−/− mice and confirmed the progressive ablation of the satellite-cell lineage in multiple muscle groups. Small numbers of Pax7-deficient cells do survive in the satellite cell position, but they arrest and die upon entering mitosis. Pax7−/− muscles are small, the fibers contain about half the normal number of nuclei, and fiber diameters are significantly reduced. Together, these data confirm an essential role for Pax7 in regulating the myogenic potential of satellite cells.
Transcription factors, including Pax7, typically recruit cofactors to facilitate appropriate regulation of target genes. However, it is not clear that Pax7 is acting simply as a transcriptional activator. Similar to Pax3, transfected Pax7 has a very modest ability to transactivate reporters in transient transfection assays. Therefore, an interesting possibility is that binding of Pax7 does not directly activate transcription, but instead promotes myogenic progression by remodeling chromatin over target promoters and possibly over larger chromosomal domains. Transcription activation would occur subsequently, after either the binding of additional transcription factors to newly accessible sites or the recruitment of additional coregulators to the Pax7 transcriptional complex.
To elucidate Pax7 function, we identified constituent proteins of the Pax7 transcriptional complex or complexes in primary myoblasts. Arguably, the best experimental method for systematically identifying protein-protein interactions involves expressing a tagged protein at its normal concentration in vivo to avoid nonphysiological interactions. Expression of the tagged protein at high levels after transient or stable transfection typically results in copurification of large amounts of chaperones. In yeast, normal levels of expression are best obtained by introducing the tag into the gene by homologous recombination. In contrast to immunoprecipitation with a single antibody, by using two affinity tags—for example, protein A and a calmodulin-binding peptide placed at the C-termini of yeast proteins (the "TAP" method)—all but the least abundant protein complexes can be purified to homogeneity.
Therefore, to molecularly dissect Pax7 function, we purified Pax7-interacting proteins using a 6x-histidine/TEV cleavage/3xFLAG tandem affinity purification (TAP) tag fused to the C-terminus of full-length mouse Pax7 and expressed this construct (Pax7-CTAP) in myoblasts by retroviral infection at physiological levels, with cultures expressing only the tag (HisFLAG-tag) serving as controls.
We performed large-scale TAPs to identify interacting proteins via MALDI-TOF mass spectrometry of silver-stained SDS-PAGE bands. This analysis resulted in the identification of a complex mixture of largely uncharacterized interacting proteins, as well as the target protein Pax7. Importantly, we identified the WD40-domain-containing protein Wdr5 as a Pax7-associated protein. The observation that Pax7 co-isolated with Wdr5 was intriguing, given that Wdr5, the trithorax-group protein Ash2L, and RbBP5 are the three common components of all histone methyltransferase (HMT) complexes that methylate histone H3 at lysine 4 (H3K4). Extensive biochemical and structural studies have revealed that Wdr5 is crucial because it associates directly with the amino acid tail of histone H34,22-25, while Ash2L, RbBP5, and Wdr5 together create the core structural platform for subsequent protein complex assembly. This suggested that Pax7 interacts with this core complex to recruit HMT complexes to target promoters in order to inscribe epigenetic modifications.
Using antibodies to the endogenous Pax7, Wdr5, and Ash2L proteins, reciprocal co-immunoprecipitation analysis revealed that antibodies reactive to Pax7 specifically co-precipitated Wdr5 and Ash2L. In addition, antibodies reactive to Wdr5 co-precipitated Pax7 and Ash2L, and antibodies reactive to Ash2L co-precipitated Pax7 and Wdr5. These results strongly support the assertion that the three proteins are found in the same complex. Together these data confirmed that, in primary myoblasts, Wdr5 and Ash2L interact with Pax7; they also support an association of Pax7 with an HMT complex.
To identify the methyltransferase responsible for this activity, we performed co-immunoprecipitations with Pax7 and blotted for the members of the MLL family of HMTs. The MLL proteins were strong candidates for this activity because of their known associations with the Wdr5-Ash2L-RbBP5 core complex. Indeed, Pax7 interacted with MLL2 but not MLL1, indicating that MLL2 is a specific HMT recruited to this complex.
To directly confirm that the Pax7-Wdr5-Ash2L-MLL2 complex possessed methyltransferase activity, we incubated Pax7 complexes with core histones in the presence of the tritiated methyl donor S-adenosyl-methionine (SAM). Histones incubated with the Pax7 immunoprecipitated-complex showed a significantly higher level of tritium incorporation than in an IgG control, demonstrating that the Pax7-Wdr5-Ash2L complex does indeed possess robust HMT activity.
Modifications of the histone tail—with regard to the amino acids in question, the order in which they are modified, and the extent of the modifications—have a significant deterministic effect on the nature of gene expression. Methylation of H3K4 marks chromatin in a conformation permissive for transcription, while trimethylated H3K4 is restricted to the 5′ promoter and coding regions and is considered a definitive marker of active genes. Experimental findings demonstrate that the Pax7-Wdr5-Ash2L-MLL2 complex specifically directs methylation of H3K4.
Taken together, these data indicate that specific binding of Pax7 to dimethylated H3K4 regulatory elements in target genes leads to the recruitment of HMT core complexes, strong H3K4 trimethylation of their regulatory elements, and ensuing activation of gene expression. This is consistent with structural data showing an interaction between the Wdr5-Ash2L core complex and histone H3, and answers the question as to how this generic complex is recruited to specific genes. They also corroborate other genomic data indicating that recruitment of this complex and subsequent trimethylation (in the 5′ promoter and coding regions) result in target gene activation. Finally, they define a specific role and molecular mechanism for Pax7 in myogenic stem cells in regulating Myf5 and other target genes. Furhermore, these experiments indicate that Pax7 enforces satellite cell commitment by recruiting an HMT complex to Myf5, resulting in transcriptional activation. Notably, the other members of the Pax gene family are essential for the embryonic specification of diverse tissues. Future experiments will establish whether they also recruit HMT complexes to target genes, thereby seeding lineage-specific gene expression programs during development.
Last updated December 2008
