AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs

Abstract

AFF1 and AFF4 belong to the AFF (AF4/FMR2) family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b (P-TEFb) and ELL1/2, in super elongation complexes (SECs). Both AFF1 and AFF4 regulate gene transcription through elongation and chromatin remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells (MSCs) is unknown. In this study, we show that small interfering RNA (siRNA)-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFF1 regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFF1-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.

Acetylation of histone H3K27 signals the transcriptional elongation for estrogen receptor alpha

“…These complexes were classified by different scaffold proteins, and they all share the common subunits such as P-TEFb and acetylated histone binding proteins AF9/ENL. Although AFF1 and AFF4 were initially identified as heterodimers and both were components of SEC in pull-down assays, they have been reported to regulate discrete subsets of target genes, and display very different affinity with their associated transcription factors [38][39][40] . Therefore, AFF1 and AFF4 may form individual SECs and the genespecificity of each SEC complex is likely determined by its associated proteins.…”

Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

“…For alizarin red staining (ARS) staining, cells were fixed after 10 days induction of osteogenic differentiation with 4% polyoxymethylene for 15 min and stained with 1% Alizarin red S (pH 4.2, Sigma-Aldrich) for 5 min. Mineralized matrix stained with alizarin red were destained with 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0), and the calcium concentration was determined by a spectrophotometer (Thermo Fisher Scientific) at 562 nm using a standard calcium curve in the same solution 62 , 63 .…”

Mesenchymal Stem Cell-Based Immunomodulation: Properties and Clinical Application

“…Currently, MSCs are regarded as a potential new therapy for a variety of human diseases. Recently, studies have focused on regulation of MSC fate with respect to their pluripotency and differentiation to promote regenerative therapeutic development [ 15 , 16 ]. Increasing numbers of clinical trials are reporting the success of MSC-based immunomodulation based on the measurement of soluble secretors and their interaction with immune cells [ 17 ].…”