Pax7 pioneer factor action requires both paired and homeo DNA binding domains.

The pioneer transcription factor Pax7 contains two DNA binding domains (DBD), a paired and a homeo domain. Previous work on Pax7 and the related Pax3 showed that each DBD binds a cognate DNA sequence, thus defining two targets of binding and possibly modalities of action. Genomic targets of Pax7 pioneer action leading to chromatin opening are enriched for composite DNA target sites containing juxtaposed sites for both paired and homeo domains. The present work investigated the implication of the DBDs in pioneer action. We show that the composite sequence is a higher affinity binding site and that efficient binding to this site involves both DBDs of the same Pax7 molecule. This binding is not sensitive to cytosine methylation of the DNA sites consistent with pioneer action within nucleosomal heterochromatin. Introduction of single amino acid mutations in either paired or homeo domain that impair binding to cognate DNA sequences showed that both DBDs must be intact for pioneer action. In contrast, only the paired domain is required for low affinity binding of heterochromatin sites. Thus, Pax7 pioneer action on heterochromatin requires unique protein:DNA interactions that are more complex compared to its simpler DNA binding modalities at accessible enhancer target sites.

Generation of PAX7 Reporter Cells to Investigate Skeletal Myogenesis from Human Pluripotent Stem Cells.

This protocol describes the use of CRISPR/Cas9-mediated homology-directed recombination to construct a PAX7-GFP reporter in human pluripotent stem cells (hPSCs). PAX7 is a key transcription factor and regulator of skeletal muscle stem/progenitor cells. We obtained heterozygous knockin reporter cells and validated their PAX7 expression using both artificial activation by the CRISPR/dCas9-VPR system and physiological activation during hPSC myogenic differentiation. These cells can serve as tools for better understanding of in vitro hPSC myogenesis and enriching myogenic cells for downstream analysis. For complete details on the use and execution of this protocol, please refer to Xi et al. (2017) and Xi et al. (2020).

DNA aptamers against the DUX4 protein reveal novel therapeutic implications for FSHD.

Aberrant expression of the transcription factor double homeobox protein 4 (DUX4) can lead to a number of diseases including facio-scapulo-humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and sarcomas. Inhibition of DUX4 may represent a therapeutic strategy for these diseases. By applying Systematic Evolution of Ligands by EXponential Enrichment (SELEX), we identified aptamers against DUX4 with specific secondary structural elements conveying high affinity to DUX4 as assessed by fluorescence resonance energy transfer and fluorescence polarization techniques. Sequences analysis of these aptamers revealed the presence of two consensus DUX4 motifs in a reverse complementary fashion forming hairpins interspersed with bulge loops at distinct positions that enlarged the binding surface with the DUX4 protein, as determined by crystal structure analysis. We demonstrate that insertion of specific structural elements into transcription factor binding oligonucleotides can enhance specificity and affinity.

Characterization of the promoter region of the bovine SIX1 gene: Roles of MyoD, PAX7, CREB and MyoG.

The SIX1 gene belongs to the family of six homeodomain transcription factors (TFs), that regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and mediate skeletal muscle growth and regeneration. Previous studies have demonstrated that SIX1 is positively correlated with body measurement traits (BMTs). However, the transcriptional regulation of SIX1 remains unclear. In the present study, we determined that bovine SIX1 was highly expressed in the longissimus thoracis. To elucidate the molecular mechanisms involved in bovine SIX1 regulation, 2-kb of the 5' regulatory region were obtained. Sequence analysis identified neither a consensus TATA box nor a CCAAT box in the 5' flanking region of bovine SIX1. However, a CpG island was predicted in the region -235 to +658 relative to the transcriptional start site (TSS). An electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay in combination with serial deletion constructs of the 5' flanking region, site-directed mutation and siRNA interference demonstrated that MyoD, PAX7 and CREB binding occur in region -689/-40 and play important roles in bovine SIX1 transcription. In addition, MyoG drives SIX1 transcription indirectly via the MEF3 motif. Taken together these interactions suggest a key functional role for SIX1 in mediating skeletal muscle growth in cattle.

The duplicated paired box protein 7 (pax7) genes differentially transcribed during Japanese flounder (Paralichthys olivaceus) embryogenesis.

PAX are important regulators of developmental processes. PAX7 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest (NC), and skeletal muscle. Here, we identified six spliced isoforms of pax7a and one pax7b and characterized their expression patterns. All of flounder Pax7a-1, Pax7a-2, Pax7a-3, and Pax7b contain a conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). However, the PD of Pax7a-4 and the HD of Pax7a-5 are not intact, and there is no HD in Pax7a-4 and Pax7a-6. pax7a and pax7b show distinct spatiotemporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that the expression patterns of pax7a and pax7b are overlapping but distinguishable in the dorsal central nervous system. pax7a is expressed in most part of the brain and the neural tube, while pax7b is expressed exclusively in the diencephalon and the midbrain. In addition, pax7a is also expressed in the cranial NC and the trunk NC. RT-PCR results show that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated pax7 genes. The duplicated pax7 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis.

Long-duration muscle dedifferentiation during limb regeneration in axolotls.

Although still debated, limb regeneration in salamanders is thought to depend on the dedifferentiation of remnant tissue occurring early after amputation and generating the progenitor cells that initiate regeneration. This dedifferentiation has been demonstrated previously by showing the fragmentation of muscle fibers into mononucleated cells and by revealing the contribution of mature muscle fibers to the regenerates by using lineage-tracing studies. Here, we provide additional evidence of dedifferentiation by showing that Pax7 (paired-box protein-7) transcripts are expressed at the ends of remnant muscle fibers in axolotls by using in situ hybridization and by demonstrating the presence of Pax7+ muscle-fiber nuclei in the early bud and mid-bud stages by means of immunohistochemical staining. During the course of regeneration, the remnant muscles did not progress; instead, muscle progenitors migrated out from the remnants and proliferated and differentiated in the new tissues at an early stage of differentiation. The regenerating muscles and remnant muscles were largely disconnected, and this left a gap between them until extremely late in the late stage of differentiation, at which point the new and old muscles connected together. Notably, Pax7 transcripts were detected in the regions of muscles that faced these gaps; thus, Pax7 expression might indicate dedifferentiation in the remnant-muscle ends and partial differentiation in the regenerating muscles. The roles of this long-duration dedifferentiation in the remnants remain unknown. However, the results presented here could support the hypothesis that long-duration muscle dedifferentiation facilitates the connection and fusion between the new and old muscles that are both in an immature state; this is because immature Pax7+ myoblasts readily fuse during developmental myogenesis.

Ret finger protein mediates Pax7-induced ubiquitination of MyoD in skeletal muscle atrophy.

Skeletal muscle atrophy results from the net loss of muscular proteins and organelles and is caused by pathologic conditions such as nerve injury, immobilization, cancer, and other metabolic diseases. Recently, ubiquitination-mediated degradation of skeletal-muscle-specific transcription factors was shown to be involved in muscle atrophy, although the mechanisms have yet to be defined. Here we report that ret finger protein (RFP), also known as TRIM27, works as an E3 ligase in Pax7-induced degradation of MyoD. Muscle injury induced by sciatic nerve transection up-regulated RFP and RFP physically interacted with both Pax7 and MyoD. RFP and Pax7 synergistically reduced the protein amounts of MyoD but not the mRNA. RFP-induced reduction of MyoD protein was blocked by proteasome inhibitors. The Pax7-induced reduction MyoD was attenuated by RFP siRNA and by MG132, a proteasome inhibitor. RFPΔR, an RFP construct that lacks the RING domain, failed to reduce MyoD amounts. RFP ubiquitinated MyoD, but RFPΔR failed to do so. Forced expression of RFP, but not RFPΔR, enhanced Pax7-induced ubiquitination of MyoD, whereas RFP siRNA blocked the ubiquitination. Sciatic nerve injury-induced muscle atrophy as well the reduction in MyoD was attenuated in RFP knockout mice. Taken together, our results show that RFP works as a novel E3 ligase in the Pax7-mediated degradation of MyoD in response to skeletal muscle atrophy.

SUMOylation of Pax7 is essential for neural crest and muscle development.

Regulatory transcription factors of the Pax family play fundamental roles in the function of multipotent cells during vertebrate development, post-natal regeneration, and cancer. Pax7 and its homologue Pax3 are important players in neural crest and muscle development. Both genes are coexpressed in various tissues and are thought to provide similar, but not identical, functions. The mechanisms that allow specific regulation of Pax7 remain largely unknown. Here, we report for the first time that Pax7 is regulated by SUMOylation. We identify the interaction of Pax7 with Ubc9, the SUMO conjugating enzyme, and reveal that SUMOylation machinery is enriched in neural crest precursors and plays a critical role in NC development. We demonstrate that Pax7 becomes SUMOylated and identify an essential role for lysine 85 (K85) in Pax7-SUMOylation. Despite high conservation surrounding K85 amongst Pax genes, we were unable to identify SUMOylation of other Pax proteins tested, including Pax3. Using a non-SUMOylatable Pax7 variant (K85 X R), we demonstrate that SUMOylation is essential for the function of Pax7 in neural crest development, C2C12 myogenic differentiation, and transcriptional transactivation. Our study provides new mechanistic insight into the molecular regulation of Pax7's function by SUMOylation in neural crest and muscle development.

Carm1 regulates Pax7 transcriptional activity through MLL1/2 recruitment during asymmetric satellite stem cell divisions.

In skeletal muscle, asymmetrically dividing satellite stem cells give rise to committed satellite cells that transcribe the myogenic determination factor Myf5, a Pax7-target gene. We identified the arginine methyltransferase Carm1 as a Pax7 interacting protein and found that Carm1 specifically methylates multiple arginines in the N terminus of Pax7. Methylated Pax7 directly binds the C-terminal cleavage forms of the trithorax proteins MLL1/2 resulting in the recruitment of the ASH2L:MLL1/2:WDR5:RBBP5 histone H3K4 methyltransferase complex to regulatory enhancers and the proximal promoter of Myf5. Finally, Carm1 is required for the induction of de novo Myf5 transcription following asymmetric satellite stem cell divisions. We defined the C-terminal MLL region as a reader domain for the recognition of arginine methylated proteins such as Pax7. Thus, arginine methylation of Pax7 by Carm1 functions as a molecular switch controlling the epigenetic induction of Myf5 during satellite stem cell asymmetric division and entry into the myogenic program.

Reciprocal inhibition between Pax7 and muscle regulatory factors modulates myogenic cell fate determination.

Postnatal growth and regeneration of skeletal muscle requires a population of resident myogenic precursors named satellite cells. The transcription factor Pax7 is critical for satellite cell biogenesis and survival and has been also implicated in satellite cell self-renewal; however, the underlying molecular mechanisms remain unclear. Previously, we showed that Pax7 overexpression in adult primary myoblasts down-regulates MyoD and prevents myogenin induction, inhibiting myogenesis. We show that Pax7 prevents muscle differentiation independently of its transcriptional activity, affecting MyoD function. Conversely, myogenin directly affects Pax7 expression and may be critical for Pax7 down-regulation in differentiating cells. Our results provide evidence for a cross-inhibitory interaction between Pax7 and members of the muscle regulatory factor family. This could represent an additional mechanism for the control of satellite cell fate decisions resulting in proliferation, differentiation, and self-renewal, necessary for skeletal muscle maintenance and repair.

Cattle cloned from increasingly differentiated muscle cells.

It has been postulated that mammalian nuclear transfer (NT) cloning efficiency is inversely correlated with donor cell differentiation status. To test this hypothesis, we compared genetically identical and increasingly differentiated donors within the myogenic lineage. Bovine male fetal muscle cells were cultured for 1-6 days in vitro. The proportion of cells displaying the following antigens was quantified by immunofluorescence microscopy: MYOD1, MYF5, PAX7, MYOG, DES, MYH, and 5-Bromo-2-deoxyuridine. Based on the antigen profile of both bulk populations and individually size-selected cells prepared for NT, donors serum-starved for 1, 4, and 5 days were classified as myogenic precursors (MPCs), myotubes (MTs), and muscle-derived fibroblasts (MFs) with purities of 92%, 85%, and 99%, respectively. Expression of the following transcripts was measured by RT-PCR in 1) cells selected for NT, 2) metaphase II oocytes, 3) NT couplets, 4) NT reconstructs, 5) NT two-cell embryos, and 6) NT blastocysts: MYOD1, MYF5, PAX7, MYOG, MYF6, ACTB, and 18S rRNA. Muscle-specific genes were silenced and remained undetectable up to the blastocyst stage, whereas housekeeping genes 18S and ACTB continued to be expressed. Differentiation status affected development to transferable embryos (118 [23%] of 520 vs. 93 [11%] of 873 vs. 66 [38%] of 174 for MPC vs. MT vs. MF, respectively, P < 0.001). However, there were no significant differences in pregnancy rate and development to weaning between the cell types (pregnancy rate: 14 [64%] of 22 vs. 8 [35%] of 23 vs. 10 [45%] of 22, and development: 4 [18%] of 22 vs. 2 [9%] of 23 vs. 3 [14%] of 22 for MPC vs. MT vs. MF, respectively).

Characterisation and expression of the paired box protein 7 (Pax7) gene in polymorphic Arctic charr (Salvelinus alpinus).

Arctic charr (Salvelinus alpinus L.) from Lake Thingvallavatn, Iceland occur as four distinct morphs: large benthivorous (LB), dwarf benthivorous (DB), piscivorous (PI) and planktonivorous (PL). The morphs differ with respect to body size, head morphology, growth rate, and life history. The aim of this study was to investigate the paired box protein 7 (Pax7) gene as a candidate for such polymorphisms due to its importance in cranio-facial, skeletal muscle, and central nervous system development. No variation in coding and intronic sequences was found between morphs. We identified 10 alternate Pax7 isoforms with insertions/deletions: a four-residue (GNRT) deletion, a GEASS insertion truncated by the first serine residue (GEAS), and a thirteen-residue insertion (GQYA/TGPEYVYCGT). The latter insertion with a threonine (T) contains a putative casein kinase II (CK-2) phosphorylation site. Pax7 spatial expression patterns were identical in embryos of DB-, LB-, and PL-morphs, and were similar to those described for zebrafish Pax7c, but a difference in temporal expression for segmentation was observed between DB and LB morphs. At the end of segmentation, novel expression was observed in the mandibular region as two bilateral domains. The potential role of multiple alternative splicing of the Pax7 gene for the generation of different Arctic charr morphs is briefly discussed.