BMP2-dependent gene regulatory network analysis reveals Klf4 as a novel transcription factor of osteoblast differentiation

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.

Chromatin Accessibility Predetermines Odontoblast Terminal Differentiation

“…They reported significant upregulation of Dmp1 , Dspp ( Chen et al, 2009 ), and Nestin ( Nes ) ( Kaukua et al, 2014 ), which are marker genes mainly expressed in the odontoblast layer [ Nes is also expressed in the pericytes in dental pulp ( Yianni and Sharpe, 2018 )]. However, the same medium could also potentially induce osteogenic differentiation when bone marrow-derived mesenchymal cells (BMSCs) are used ( Yu et al, 2021 ). Thus, whether this model can mimic in vivo odontoblast differentiation remains unclear.…”

Emerging Single-Cell Technological Approaches to Investigate Chromatin Dynamics and Centromere Regulation in Human Health and Disease

“…For instance, the genomic profiling of native R loops, which plays a pivotal function in many biological processes with consequences in human disease [116]. Yu et al have employed the H3K27Ac CUT&Tag protocol to investigate BMP-dependent active enhancers during osteoblast differentiation [117]. Bartosovic et al used CUT&TAG to profile histone modifications and transcription factors occupancy at the single-cell level in the mouse brain [118].…”

Identification of chromatin states during zebrafish gastrulation using CUT&RUN and CUT&Tag

“…However, compared to CUT&RUN, current protocols may have more limited effectiveness at detecting transient protein‐DNA interactions involving transcription factors, and in some cases, the use of Tn5 transposase could potentially bias data toward open chromatin 38,39 . Thus far, CUT&Tag has been applied to assay enrichment of modified histones in plant and mammalian tissues 38,40‐45 …”

“…38,39 Thus far, CUT&Tag has been applied to assay enrichment of modified histones in plant and mammalian tissues. 38,[40][41][42][43][44][45] Here we describe successful implementation of CUT&-RUN and CUT&Tag methods for profiling protein-DNA interactions in zebrafish embryos. Zebrafish have long been recognized as a powerful system for the study of early vertebrate development, as external fertilization facilitates molecular analysis at the earliest stages of embryogenesis.…”