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Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [ChIP-Seq 2]

ABSTRACT: To identify Sp7’s molecular interactions in vivo, we used gene-targeting strategies to generate an Sp7-Biotin-3xFLAG knock-in mouse (Sp7-BioFL mouse). This approach appends a biotin (Bio) recognition motif and three copies of the FLAG (FL) epitope at the C-terminus of the Sp7 protein. To validate this knockin system, we compared ChIP-seq results in the MC3T3E1 osteoblast cell line, introducing Sp7 forms that were epitope-tagged at either the N- (N-terminal FLAG tag; MC3_FLAG-Sp7) or C-terminus (C-terminal Biotin-FLAG tag, as in the in vivo targeted allele; MC3_Sp7-BioFLAG). These data show an extensive overlap, suggesting that FLAG tagging at different positions gives comparable outcomes. Second, we examined ChIP on wild-type calvarial primary osteoblasts (POB) using an anti-Sp7 antibody (POB_Sp7Ab-ChIP). The majority of Sp7-Ab ChIP-seq peaks (95%) overlapped with the larger set identified by Sp7-BioFL ChIP-seq.

ORGANISM(S): Mus musculus

PROVIDER: GSE80231 | GEO | 2016/04/28

SECONDARY ACCESSION(S): PRJNA318364

REPOSITORIES: GEO

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Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [ChIP-Seq]

Project description:Sp7/Osterix is a master regulator of osteoblast specification. To identify the Sp7-mediated gene regulatory network in osteoblasts, we performed Sp7 ChIP-seq on primary mouse calvarial osteoblasts comparing the DNA binding profile with the transcriptional profile of Sp7-positive osteoblasts. Analysis of these identified a network of Sp7 regulated osteoblast targets and provides a new insight into the mode of Sp7 action in osteoblast. To further study for the Sp7 mode, we performed ChIP-seq for Sp1, Sp7, Dlx5, as a potential Sp7 partner identified in this study and mutated Sp7 which has mutations in the zinc finger domain, by using in vitro system with a pre-osteoblast mouse cell line, MC3T3E1.

2016-04-28 | GSE76185 | GEO

Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [RNA-Seq]

Project description:Sp7/Osterix is a master regulator of osteoblast specification. To identify transcripts profile in Sp7 positive osteoblast, we performed RNA-seq on primary mouse calvarial cells obtained from Sp7-GFP reporter mice at P1. By hierarchical clustering using transcriptional profiles for chondrocytes and mouse embryonic fibroblasts (MEFs) together with the osteoblast data, we identified cell-type enriched gene expression signatures in osteoblasts, chondrocytes and MEFs. In conjunction with Sp7 ChIP-seq in osteoblast, we identified putative Sp7 targets which underlie the osteoblast regulatory program.

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Project description:SP7/Osterix is a transcription factor critical for osteoblast maturation and bone formation. We identidied a missense variant (c.926C>G:p.S309W) in SP7 in a patient with a unique high turnover bone disease. Mice with the corresponding variant similarly showed a complex skeletal phenotype distinct from that of Sp7-null mice. We therefore performed ChIP-Seq in primary chondrocytes to study how the mutation alters the genomic binding of SP7.

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Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 13 Time Points (0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates. ChIPs comparing the occupancy of Rap1 in a strain containing two copies of Rap1, one with a Flag tag and one with a Myc tag, and expressed from an identical promoter.

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SP7 mutant S309W genomic binding in MC3T3 cells

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Project description:Some cuboidal osteoblasts differentiate into bone-embedded, dendrite-bearing osteocytes through the poorly-understood process of osteocytogenesis. Here, we report that the transcription factor Sp7 plays an essential role in osteocytogenesis. Severe defects in bone integrity and osteocyte dendrite morphology are noted in mice lacking Sp7 at the stage of the osteoblast-to-osteocyte transition. In osteocytes, Sp7 controls expression of a neuronally-enriched gene network. Analysis of the osteocyte-specific Sp7 cistrome reveals distinct genomic binding motifs and target sites distinct from those in osteoblasts. Amongst osteocyte-specific Sp7 targets, the secreted peptide osteocrin rescues Sp7-deficient defects. Single-cell transcriptional profiling of cells undergoing osteocytogenesis identifies novel Sp7-dependent transitional cell types enriched in genes linked to human fracture risk. Finally, humans with an SP7 R316C mutation display osteocyte morphology defects similar to those observed in mouse models. These findings demonstrate that cuboidal osteoblasts use a neuronally-enriched Sp7/osteocrin gene expression program to differentiate into dendrite-bearing osteocytes.

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