Loss of β‐Actin Leads to Accelerated Mineralization and Dysregulation of Osteoblast‐Differentiation Genes during Osteogenic Reprogramming

Abstract

Actin plays fundamental roles in both the cytoplasm and the cell nucleus. In the nucleus,-actin regulates neuronal reprogramming by consolidating a heterochromatin landscape required for transcription of neuronal gene programs, yet it remains unknown whether it has a role in other differentiation models. To explore the potential roles of-actin in osteogenesis,-actin wild-type (WT) and-actin knockout (KO) mouse embryonic fibroblasts (MEFs) are reprogrammed to osteoblast-like cells using small molecules in vitro. It is discovered that loss of-actin leads to an accelerated mineralization phenotype (hypermineralization), accompanied with enhanced formation of extracellular hydroxyapatite microcrystals, which originate in the mitochondria in the form of microgranules. This phenotype is a consequence of rapid upregulation of mitochondrial genes including those involved in oxidative phosphorylation (OXPHOS) in reprogrammed KO cells. It is further found that osteogenic gene programs are differentially regulated between WT and KO cells, with clusters of genes exhibiting different temporal expression patterns. A novel function for-actin in osteogenic reprogramming through a mitochondria-based mechanism that controls cell-mediated mineralization is proposed.

β-actin dependent chromatin remodeling mediates compartment level changes in 3D genome architecture

“…All expression levels of target genes were normalized to the expression of Nono reference gene. qPCR analysis of DMP1, SPP1, and SDHA was performed as previously described 40 , 55 . The primers used for qPCR analysis are included in supplementary table 2…”

Regulation of nuclear actin levels and MRTF/SRF target gene expression during PC6.3 cell differentiation

“…Actin could be one possible candidate to regulate these global changes in chromatin organization during differentiation. Indeed, mouse embryonic fibroblasts (MEFs) lacking the β-actin gene display altered 3D genome architecture (Mahmood et al, 2021) and are defective in their reprogramming capacity towards osteoblast-like cells (Gjorgjieva et al, 2020), adipocytes (Al-Sayegh et al, 2020) and chemical-induced neurons. In this latter case, β-actin deficiency is linked to loss of chromatin-binding of Brg1, an ATPase of the BAF chromatin remodeling complex.…”

The Use of Stem Cell-Derived Organoids in Disease Modeling: An Update

“…Laminin, a component of the basement membrane [ 150 ], recapitulates the ICM niche preventing the polarization of the blastomeres into ICM and trophectoderm [ 149 ]. The first method for successfully establishing hESCs lines was to co-culture isolated stem cells with mouse embryonic fibroblasts (MEFs), which also have differentiation capabilities to other cell types [ 151 , 152 , 153 , 154 ]). As reported by Thompson et al, the hESCs were grown on MEFs feeders in a medium containing fetal bovine serum.…”