Abstract
The promyelocytic leukemia protein (PML) is expressed in most normal human tissues and forms nuclear bodies (NBs) that have roles in gene regulation and cellular processes such as DNA repair, cell cycle control, and cell fate decisions. Using murine C2C12 myoblasts, we demonstrate that activation of skeletal muscle differentiation results in loss of PML and PML NBs prior to myotube fusion. Myotube formation was associated with marked chromatin reorganization and the relocalization of DAXX from PML NBs to chromocentres. MyoD expression was sufficient to cause PML NB loss, and silencing of PML induced DAXX relocalization. Fusion of C2C12 cells using the reptilian reovirus p14 fusogenic protein failed to disrupt PML NBs yet still promoted DAXX redistribution and loss; whereas ectopic expression of PML in differentiated cells only partially restored PML NB formation and DAXX localization at NBs. Finally, we determined that the C-terminal SUMO-interacting motif of DAXX is required for its colocalization with ATRX in heterochromatin domains during myotube formation. These data support a model in which activation of myogenic differentiation results in PML NB loss, chromatin reorganization and DAXX relocalization, and provides a paradigm for understanding the consequence of PML loss in other cellular contexts, such as during cancer development and progression.
Highlights
Identified and characterized as a tumor suppressor, the promyelocytic leukemia (PML) protein has an important role in the regulation of gene expression.[1]
In order to investigate the role of PML nuclear bodies (PML NBs) in myogenesis we used the C2C12 mouse muscle myoblast cell line, where substituting mitogen-rich FBS with 2% mitogen-poor horse serum can induce their differentiation into multinucleated myotubes
As previous studies indicate that lack of PML and PML NBs results in redistribution of constitutive PML NB components such as DAXX,[2,30] we examined DAXX localization in C2C12 myoblasts and myotubes (Figure 2a)
Summary
Identified and characterized as a tumor suppressor, the promyelocytic leukemia (PML) protein has an important role in the regulation of gene expression.[1]. SUMO modification of PML is important for PML NB formation and recruitment of SUMO-interacting motif (SIM)-containing PML NB components such the histone chaperone DAXX.[2,3] PML NBs associate with over 150 different cellular proteins,[4,5] either constitutively such as transcriptional repressors SP100 and DAXX,[2,6] or transiently in response to stress such as the DNA repair factors MRE11 and TOPBP1.7–9 The majority of these proteins are involved in regulating gene expression as transcription factors (e.g., STAT3, SP1, GATA2), transcription factor modifying enzymes (e.g., PIAS1, SENP1, HIPK2) or chromatin modifiers (e.g., DAXX, CBP, HDACs).[4,5] . The coordinated execution of wellcontrolled gene expression programs are required to establish specific cell and tissue types, and a role for nuclear structure in this process is only being explored. One such process is skeletal muscle development. Using the Biology, Dalhousie University, Halifax, NS, Canada, B3H 4R2
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