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Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin [scRNA-Seq]

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE154718 | GEO | 2021/09/20

REPOSITORIES: GEO

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Project description:Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-seq analyses, we show that while substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast-like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by ChIP-seq, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)2D3 that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by RXR and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)2D3-regulated genes, the expression of others adjacent to VDR binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)2D3 was found to induce the Boc and Cdon co-receptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)2D3. ChIP-seq was performed on differentiated IDGSW3 cells at day 35 for VDR and RXR following 3 hour vehicle or 1,25(OH)2d3 treatment in biological replicate. ChIP-seq was also performed for H3K4me1, H3K4me2, H3K4me3, H3K27Ac, H4K5Ac, H3K9Ac, H4K20me1, H3K36me3, or H3K9me3 at day 3 and day 35 of differentiation in the basal condition.

Project description:Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-seq analyses, we show that while substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast-like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by ChIP-seq, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)2D3 that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by RXR and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)2D3-regulated genes, the expression of others adjacent to VDR binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)2D3 was found to induce the Boc and Cdon co-receptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)2D3. Two sets of IDG-SW3 differentitiation mRNA-seq experiments are presented here in biological triplicate. One a differentiation time course in the basal condition at days 3, 7, 14, 21, 28, 35. The second experiment is IDG-SW3 cells differentiated to day 3 or day 35 and treated with vehicle or 100nM 1,25(OH)2D3 for 24 hours prior to mRNA isolation and sequencing.

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Control of osteocyte dendrite formation by Sp7 and its target gene osteocrin [scRNA-Seq]

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