Purα and Purβ Collaborate with Sp3 To Negatively Regulate β-Myosin Heavy Chain Gene Expression duringSkeletal Muscle Inactivity

Abstract

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased ␤-myosin heavy chain (␤MyHC) gene expression. Our previous work showed that Sp3 contributes to decreased ␤MyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Pur␣, and Pur␤ proteins mediate repression of ␤MyHC expression under NWB conditions. Binding of Pur␣ or Pur␤ to the single-stranded ␤MyHC distal negative regulatory element-sense strand (d␤NRE-S) element is markedly increased under NWB conditions. Ectopic expression of Pur␣ and Pur␤ decreased ␤MyHC reporter gene expression, while mutation of the d␤NRE-S element increased expression in C2C12 myotubes. The d␤NRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Pur␣ and Pur␤ decreased endogenous Sp3 and Pur protein levels and increased ␤MyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Pur␣, Pur␤, and Sp3, while chromatin immunoprecipitation assays demonstrated Pur␣, Pur␤, and Sp3 binding to the ␤MyHC proximal promoter region harboring the d␤NRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

A Bifunctional Intronic Element Regulates the Expression of the Arginine/Lysine Transporter Cat-1 via Mechanisms Involving the Purine-rich Element Binding Protein A (Purα)

“…13). Consensus binding sites for the protein are (GGN) n Ͼ(GGNN) n Ͼ(GGNNN) n , where N is not a G. Pur␣ also regulates transcription via GA/GC-rich regions with the cooperative action of Sp1 and Sp3 (14,21). There is one report that Pur␣ inhibits cAMP-response element-mediated transcription by binding to a purine-rich element adjacent to the cAMP-response element binding site (27).…”

“…ATF4 and CHOP antibodies were from Santa Cruz Biotechnology. Pur␣ antibodies were from Dr. R. J. Kelm (20,21). Immunoprecipitated and purified DNA fragments were analyzed by PCR and qPCR.…”

Fiber Types in Mammalian Skeletal Muscles

“…On the other hand, expression of fast contractile proteins is not activated by Sox6 overexpression, suggesting that other targets of miR-499 or miR-208b are required for activation of the fast gene program. The transcriptional repressors Pur␤ and Sp3, which were previously shown to repress MyHC-␤/slow expression in mouse skeletal muscle (381), are also targets of miR-499 and miR-208b (817). An open issue here is whether this mechanism involving muscle-specific miRNAs and Sox6, which controls fiber type diversification in developing muscle, as revealed by transgenic and knockout experiments, is also involved in the maintenance of fibre type properties in adult skeletal muscle.…”

Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism

“…fiber program by downregulating the expression of transcriptional repressors, including Sox6 and Purb, which, in turn, suppress slowtwitch genes (19,20,(32)(33)(34)(35)(36).…”