Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.
We studied Tcirg1 gene expression on RANKL-induced osteoclastic differentiation of the mouse model RAW264.7 cells. We identified a mechanism involving PARP-1 inhibition release and JunD/Fra-2 binding, which is responsible for Tcirg1 gene upregulation.


BACKGROUND
The Tcirg1 gene encodes the a3 isoform of the V-ATPase a subunit, which plays a critical role in the resorption activity of the osteoclast. Using serial deletion constructs of the Tcirg1 gene promoter, we performed a transcriptional study to identify factor(s) involved in the regulation of the RANKL-induced gene expression.


METHODS
The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cells differentiation process. We next performed sequence analysis, EMSA, UV cross-linking, qPCR, and gel supershift experiments to identify the factor(s) interacting with the promoter.


RESULTS
A deletion of the -1297-1244 region led to the disappearance of the RANKL-induced promoter activity. EMSA experiments showed the binding of two factors that undergo differential binding on RANKL treatment. Supershift experiments led us to identify the dimer JunD/Fra-2 as the binding activity associated with the -1297/-1268 Tcirg1 gene promoter sequence in response to RANKL. Moreover, we observed poly(ADP-ribose) polymerase-1 (PARP-1) binding to an adjacent site (-1270/-1256), and this interaction was disrupted after RANKL treatment.


CONCLUSIONS
We provide data that identify junD proto-oncogene (JunD) and Fos-related antigen 2 (Fra-2) as the activator protein-1 (AP-1) factors responsible for the RANKL-induced upregulation of the mouse Tcirg1 gene expression. Moreover, we identified another binding site for PARP-1 that might account for the repression of Tcirg1 gene expression in pre-osteoclastic cells.
Beranger
Guillaume E
GE
GéPITOS, Université Nice-Sophia Antipolis, CNRS, UFR de Médecine, Nice, France.
Momier
David
D
Guigonis
Jean-Marie
JM
Samson
Michel
M
Carle
Georges F
GF
Scimeca
Jean-Claude
JC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects* / Q000528:radiation effects; D003432:Cross-Linking Reagents; D019281:Dimerization; D051921:Fos-Related Antigen-2 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D051379:Mice; D008810:Mice, Inbred C57BL; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000528:radiation effects; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000528:radiation effects; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D053245:RANK Ligand / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014466:Ultraviolet Rays; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics*
C484226:Atp6ap1 protein, mouse; D003432:Cross-Linking Reagents; D051921:Fos-Related Antigen-2; D016755:Proto-Oncogene Proteins c-jun; D053245:RANK Ligand; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D025262:Vacuolar Proton-Translocating ATPases