The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle
The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.
Introduction
Postnatal muscle stem cells, namely satellite cells (SCs), allow neonatal/juvenile growth phase of normal skeletal myofibers as well as the homeostasis and repair following injury of adult skeletal muscle1,2. SCs are histologically located in a niche environment between the sarcolemma and the basal lamina of the muscle fiber. Despite heterogeneous in gene expression signature, stemness, and myogenic differentiation potential, SCs are characterized by the expression of the paired domain transcription factor Pax73. The ablation of the total pool of Pax7+ cells in adulthood results in the complete loss of muscle regeneration4,5. In adult muscle, Pax7+ cells are mitotically quiescent but quickly enter the cell cycle in response to injury and muscle degeneration to both self-renew and differentiate into functional myofibers. A dynamic interplay exists between SCs and the stem cell niche: the activity of SCs is influenced not only by intrinsic factors, but also by signals provided by the niche6. In addition, the niche has a role in the regulation of SCs asymmetric division, which depends on the relative position of daughter cells in relation to the myofiber7. Asymmetric division, which generates two unequal daughter cells, is a key mechanism that allows the maintenance of the stem cells pool and tissue homeostasis by supporting the balance between self-renewal and differentiation7.
Multiple signaling pathways and rapid expression of myogenic transcription factors (TFs) govern SCs activation. In particular, the early expression of Myf5 and MyoD in activated SCs controls the myogenic program, depending on which of the two TFs predominates: while translation of Myf5 mRNA enhances SCs activation, MyoD mainly drives the expansion of a cell population that is committed toward early differentiation3,8. After limited rounds of proliferation, the majority of Pax7+ cells begin a differentiation program characterized by MyoD-dependent activation of Myogenin and Mef2 and decrease in Pax7 levels9.
The TF NF-Y is composed by NF-YA, NF-YB, and NF-YC subunits. NF-YA is the DNA binding subunit of the heterocomplex, which specifically binds to CCAAT boxes, common regulatory DNA elements within promoters and enhancers10,11. The NF-YA gene encodes for two different splice transcripts, NF-YA long (NF-YAl) and NF-YA short (NF-YAs), the last one lacking 28/29 aminoacids within the transactivation domain. In mouse embryonic stem cells (ESCs), NF-YAs is expressed in proliferating cells and a switch to NF-YAl occurs during differentiation12.
In mature skeletal muscle, NF-YA expression is absent and, consequently, NF-Y binding to its target genes is lost13. NF-Y is a well-known positive regulator of cell proliferation via transcriptional activation of cell growth genes. Therefore, it is generally accepted that the decrease in NF-YA levels is a key step that allows the exit from the cell cycle and the activation of the myogenic differentiation program during myoblast differentiation. Indeed, transient NF-YA overexpression impairs myoblast differentiation in vitro13,14. Despite this, we recently showed that stable overexpression of NF-YAl in C2C12 myoblasts enhances the differentiation program through the activation of new identified NF-Y target genes, such as Cdkn1c (p57) and Mef2d, which are important for early muscle differentiation. Oppositely, forced expression of NF-YAs maintains the cells into a proliferative status and inhibits the differentiation program15. Recent results obtained by CRISPR-Cas9 forced deletion of exon3, retained in NF-YAl, highlighted that the switch from NF-YAl to NF-YAs interferes with the differentiation program but not with the proliferative ability of C2C12 cells16. While in wt mice muscle NF-YA is undetectable, it is expressed in the nuclei of dystrophic mdx mouse muscles, which are characterized by extensive activation of SCs to compensate myofibers degeneration. In accordance with NF-YA expression, the NF-Y heterotrimer binds the promoters of its target genes, which are upregulated in mdx muscles14. Taking into consideration these results together with data showing that NF-YA expression controls stemness and proliferation of mouse and human stem cells12,17, we decided to investigate the biology of NF-Y in muscle stem cells. Unfortunately, the ability to study NF-Y in post-natal tissues has been hampered by early embryonic lethality of NF-YA null mice18.
In this study, we used an inducible knock out mouse model to selectively disrupt NF-YA expression in Pax7+ SCs and we demonstrated that NF-Y has key functions in the regulation of muscle stem cell fate. We present evidence that NF-Y is important for adult SCs maintenance and myogenic differentiation.
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