Abstract
Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2fx/fx mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n > 6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7 + lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4fx/+ mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly “co-bound” with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7 + lineage, in which Klf4 is a novel transcription factor.
Highlights
The vertebrate skeleton is made of cartilage and bone, which is derived from three different embryonic lineages[1]
H&E staining confirmed that the trabecular bone number and the width of cortical bone were reduced in bone morphogenetic protein 2 (BMP2)-cKO (Fig. 1G)
By combining RNA-seq with ATAC-seq, we found that the loss of BMP2 in Sp7+ lineage bone marrow stromal cells (BMSCs) resulted in a decrease in the expression and accessibility of nucleosome-free regions near osteogenesis-related genes, which is responsible for the bone defects observed in BMP2-cKO
Summary
The vertebrate skeleton is made of cartilage and bone, which is derived from three different embryonic lineages[1]. Cells arising in these lineages proliferate and migrate to form mesenchymal condensations[2]. Intramembranous ossification and endochondral ossification are two major methods of mesenchymal cells involved in bone formation[3,4]. In both approaches, Official journal of the Cell Death Differentiation Association. Yu et al Cell Death and Disease (2021)12:197 mesenchyme-derived osteoblasts play a critical role in bone formation and, bone remodeling, involving a complex gene regulatory network. A clear understanding of how tissue-specific TFs interact with each other and their downstream targets will provide insights into the mechanism of gene regulation in osteoblast differentiation
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