Inhibition of Ezh2 redistributes bivalent domains within transcriptional regulators associated with WNT and Hedgehog pathways in osteoblasts.

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss.

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts.

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFß1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFß1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis.

Brd4 is required for chondrocyte differentiation and endochondral ossification.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4fl/fl: Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification.

Brd4 Inactivation Increases Adenoviral Delivery of BMP2 for Paracrine Stimulation of Osteogenic Differentiation as a Gene Therapeutic Concept to Enhance Bone Healing.

Bromodomain (BRD) proteins are histone code interpreters that recognize acetylated lysines and link the dynamic state of chromatin with the transcriptional machinery. Here, we demonstrate that ablation of the Brd4 gene in primary mouse bone marrow-derived mesenchymal stem cells via a conditional Brd4fl/fl allele suppresses osteogenic lineage commitment. Remarkably, loss of Brd4 function also enhances expression of genes in engineered adenoviral vectors, including Cre recombinase and green fluorescent protein (GFP). Similarly, vector-based expression of BMP2 mRNA and protein levels are enhanced upon Brd4 depletion in cells transduced with an adenoviral vector that expresses BMP2 (Ad-BMP2). Importantly, Brd4 depletion in MC3T3-E1 and human adipose-derived mesenchymal stem cells (AMSCs) transduced with Ad-BMP2 enhances osteogenic differentiation of naïve MC3T3-E1 cells via paracrine mechanisms based on transwell and conditioned medium studies. Our studies indicate that Brd4 depletion enhances adenoviral transgene expression in mammalian cells, which can be leveraged as a therapeutic strategy to improve viral vector-based gene therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Epigenetic Control of Osteogenic Lineage Commitment.

Within the eukaryotic nucleus the genomic DNA is organized into chromatin by stably interacting with the histone proteins as well as with several other nuclear components including non-histone proteins and non-coding RNAs. Together these interactions distribute the genetic material into chromatin subdomains which can exhibit higher and lower compaction levels. This organization contributes to differentially control the access to genomic sequences encoding key regulatory genetic information. In this context, epigenetic mechanisms play a critical role in the regulation of gene expression as they modify the degree of chromatin compaction to facilitate both activation and repression of transcription. Among the most studied epigenetic mechanisms we find the methylation of DNA, ATP-dependent chromatin remodeling, and enzyme-mediated deposition and elimination of post-translational modifications at histone and non-histone proteins. In this mini review, we discuss evidence that supports the role of these epigenetic mechanisms during transcriptional control of osteoblast-related genes. Special attention is dedicated to mechanisms of epigenetic control operating at the Runx2 and Sp7 genes coding for the two principal master regulators of the osteogenic lineage during mesenchymal stem cell commitment.