Notch Gain of Function Inhibits Chondrocyte Differentiation via Rbpj-Dependent Suppression of Sox9

저자: Shan Chen, Jianning Tao, Yangjin Bae, Ming-Ming Jiang , Terry Bertin , Yuqing Chen, Tao Yang, and Brendan Lee

저널: J Bone Miner Res. 2013 Mar;28(3):649-59

사용한 제품: Dynabeads Streptavidin MyOne T1 (Cat# 65601, 65602)

Abstract

Notch signaling plays a critical role during development by directing the binary cell fate decision between progenitors and differentiated cells. Previous studies have shown sustained Notch activation in cartilage leads to chondrodysplasia. Genetic evidence indicates that Notch regulates limb bud mesenchymal stem cell differentiation into chondrocytes via an Rbpj-dependent Notch pathway. However, it is still unknown how Notch governs chondrogenesis in the axial skeleton where Notch serves a primary patterning function. We hypothesized that both Rbpj-dependent and Rbpj-independent Notch signaling mechanisms might be involved. Cartilage-specific Notch gain-of-function (GOF) mutant mice display chondrodysplasia accompanied by loss of Sox9 expression in vertebrae. To evaluate the contribution of an Rbpj-dependent Notch signaling to this phenotype, we deleted Rbpj on the Notch GOF background. These mice showed persistent spine abnormalities characterized by ‘‘butterfly’’ vertebrae suggesting that removal of Rbpj does not fully rescue the axial skeleton deformities caused by Notch GOF. However, Sox9 protein level was restored in Rbpj-deficient Notch GOF mice compared with Notch GOF mutants, demonstrating that regulation of Sox9 expression is canonical or Rbpj-dependent. To further understand the molecular basis of this regulation, we performed chromatin immunoprecipitation (ChIP) assays and detected the recruitment of the Rbpj/ NICD transcription complex to Rbpj-binding sites upstream of the Sox9 promoter. The association of the Rbpj/NICD complex with the Sox9 promoter is associated with transcriptional repression of Sox9 in a cellular model of chondrocyte differentiation. Hence, Notch negatively regulates chondrocyte differentiation in the axial skeleton by suppressing Sox9 transcription, and Rbpj-independent Notch signaling mechanisms may also contribute to axial skeletogenesis.   2013 American Society for Bone and Mineral Research

제품 사용법

In vivo biotinylation ChIP (chromatin immunoprecipitation) assay

A biotin acceptor followed by a TEV protease cleavage site is incorporated at the 5’ end of Rbpj, Notch1 ICD (N1ICD), or Notch2 ICD (N2ICD). BirA, the E. coli biotin transferase, conjugates biotin to the biotin acceptor, which can be captured by Streptavidin beads. TEV protease is applied to separate the protein from Streptavidin beads and the ChIP assay proceeds according to the standard protocol. The cells were transfected with Piggybacbased vectors to insert three transgenes into the genome; the transgenes are rtTA (Tetracycline responsive transactivator), BirA (biotin transferase) and Rbpj, Notch2 ICD, or Notch1 ICD tagged with BTEV peptide (biotin acceptor with TEV cleavages site). A Tet-on expression system was employed to induce transcription of Rbpj or NICD with Doxycyclin (Sigma-Aldrich, D9891–25G)administration. ATDC5 cell lines were treated with Doxycycline (Sigma-Aldrich, D9891–25G) 6 hours before collection for ChIP assay. Fixation and nuclear cell lysate extraction procedures are as described before.(18) In brief, nuclear lysate was sonicated with BioruptorXL for 24 cycles; each cycle consisted of a 30-second sonication followed by a 30-second pause. Cell debris was cleared by centrifugation at 14,000 rpm for 5 minutes, and the cleared lysate was diluted 2.5 times with dilution buffer. Blocked T1 beads (100mL) were added to the sonicated lysates and further rotated overnight at 48C. Streptavidin beads (Dynabeads MyOne Streptavidin T1, Invitrogen, Carlsbad, CA, USA) were washed following standard ChIP washing conditions with four additional washes containing 1% SDS/TE at room temperature and 1% Triton X-100/TE, 0.1% Triton X-100/TE, TEV buffer at 48C. Streptavidin beads were pulled down magnetically and digested with TEV (AcTEV Protease, catalog no. 12575–015, Invitrogen) at room temperature for 2 hours. Supernatants were collected and underwent reverse cross-linking followed by DNA precipitation with phenol-chloroform. The Sox9 gene coordinate and transcription start site positions were obtained through the UCSC browser. The region 6.8 kb upstream and 0.5 kb downstream of the transcription start site was uploaded to Matinspector software from the Genomatrix software suite (an online analysis tool warehouse). After selecting Rbpj as the target transcription factor, the binding sites of Rbpj were returned by the software and located back onto genomic coordinates. Primers flanking 100 bp 5’ or 3’ to the binding sites were designed for PCR and qPCR using Primer 3 software.