RANKL treatment releases the negative regulation of the poly(ADP-ribose) polymerase-1 on Tcirg1 gene expression during osteoclastogenesis.
The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis.


BACKGROUND
The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for approximately 60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point.


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


RESULTS
The -3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to -1589 retained this response to RANKL. A deletion up to -1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the -1589/-1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression.


CONCLUSIONS
We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function.
Beranger
Guillaume E
GE
GéPITOS-K2943 CNRS/UNSA, Faculté de Médecine de l'Université de Nice-Sophia Antipolis, Nice, France.
Momier
David
D
Rochet
Nathalie
N
Quincey
Danielle
D
Guigonis
Jean-Marie
JM
Samson
Michel
M
Carle
Georges F
GF
Scimeca
Jean-Claude
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005786:Gene Expression Regulation; D051379:Mice; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D053245:RANK Ligand / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D014158:Transcription, Genetic; D025262:Vacuolar Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics*
C484226:Atp6ap1 protein, mouse; D053245:RANK Ligand; D012333:RNA, Messenger; D011065:Poly(ADP-ribose) Polymerases; D025262:Vacuolar Proton-Translocating ATPases