The role of Trithorax family regulating osteogenic and Chondrogenic differentiation in mesenchymal stem cells

Abstract Mesenchymal stem/stromal cells (MSCs) hold great promise and clinical efficacy in bone/cartilage regeneration. With a deeper understanding of stem cell biology over the past decade, epigenetics stands out as one of the most promising ways to control MSCs differentiation. Trithorax group (TrxG) proteins, including the COMPASS family, ASH1L, CBP/p300 as histone modifying factors, and the SWI/SNF complexes as chromatin remodelers, play an important role in gene expression regulation during the process of stem cell differentiation. This review summarises the components and functions of TrxG complexes. We provide an overview of the regulation mechanisms of TrxG in MSCs osteogenic and chondrogenic differentiation, and discuss the prospects of epigenetic regulation mediated by TrxG in bone and cartilage regeneration.


| INTRODUCTION
Bone is a metabolically active and dynamic tissue with the capability of rapid remodelling to heal defects smaller than critical size perfectly.
Bone regeneration is required under various physiological and pathological situations that cause bone loss including trauma, infection, skeletal abnormality or tumour. 1  There is mounting evidence suggesting that epigenetic regulation of gene expression plays an essential role in stem cell fate determination during development. 8,9 Epigenetic modifications occur on the chromatin level without changing the DNA sequence. DNA methylation and histone modifications are key epigenetic patterns that play extensive roles in gene regulation. 10 Concerning histone modification, the most studied epigenetic factors over decades are Polycomb group (PcG) and Trithorax group (TrxG) of proteins. Initially discovered in Drosophila as heterogeneous groups of factors, PcG and TrxG proteins have antagonistic roles in transcriptional regulations of homeotic (HOX) genes and other target genes. 11 The Trithorax proteins activate gene expression and counteract PcG-mediated transcriptional repression by modifying chromatin through their histone methyltransferase Chenghao Song author contributed equally. or acetyltransferase activities. They are profoundly involved in stem cell proliferation and differentiation. 12 In this review, we briefly introduce the composition, biological function, and regulation mechanisms of TrxG proteins, and discuss the regulatory role of TrxG proteins in MSCs osteogenic and chondrogenic differentiation.

| COMPASS COMPLEXES
As a core subunit of COMPASS KMTs, WRAD is required for complex assembling and functioning. Structurally, DPY30 directly binds to ASH2L via a DPY-30 binding-motif (DBM) on the C-terminus of ASH2L, while RBBP5 interacts with the SPRY domain of ASH2L and bridges it to WDR5. 23,24 The WRAD subcomplex possesses an H3K4 monomethylation activity independently and prefers histone H3/H4 tetramer instead of nucleosomal H3 as substrates. WDR5, RBBP5, and ASH2L form a minimal complex required for H3 methylation while DPY30 functions to increase complex stability, substrate specificity, and enzymatic activity. 25,26 In vitro experiments revealed that in the absence of WRAD, the SET domain of SETD1A protein is inactive and the MLL proteins merely have weak mono-or dimethyltransferases activities, suggesting that the interaction with WRAD subcomplex is crucial for COMPASS complexes canonical enzymatic functions. 27 The regulatory role of WRAD subunits, especially WDR5, on stem cells differentiation has been well investigated. WDR5 belongs to a large protein family containing the conservative WD-repeats domains and is involved in multiprotein complex assembling, localization and regulation. 16,28 The WD-repeat β-propeller structures of WDR5 interact with RBBP5 protein and the WDR5-interacting (Win) motif of the six SETdomain-containing proteins, mediating COMPASS complexes assembling. 29,30 Moreover, WDR5 can directly bind the N-terminal tail of histone H3 mediating enzyme-substrate interaction. 31  and HOXC8 promoters, associated with reduced HOX gene expression. 44 A member of HOX genes, distal-less homeobox 3 (DLX3), promotes osteogenic differentiation of stem cells by targeting RUNX2.
In vitro experiments in human dental follicle stem cells (DFCs) showed that activation of DLX3 expression was regulated by MLL1/2 complexes and SUMO specific peptidase 3 (SENP3). Decreased deposition of MLL1/2 complexes components and reduction of H3K4me3 marks were found on the DLX3 gene when SENP3 was knockdown, leading to inhibited osteogenic differentiation of DFCs. 45 Menin 1(MEN1) is considered as a tumour suppressor associated with multiple endocrine neoplasia type 1 sydrome. 46  and Bglap. 48,49 In addition, menin was co-immunoprecipitated with JUND, the activator protein-1 transcription factor subunit, when cotransfected into MC3T3-E1 cells, and suppressed JUND induced osteoblasts maturation. 51  ATPase activity of SWI/SNF is required for Bglap transcription. 82 Similarly, BRG1 and BRM were found to be enriched at the Osx promoter in osteogenic-differentiated C3H10T1/2 cells and the catalytic activity of SWI/SNF is required for Osx expression. 83 However, the transcription activation of Runx2/p57 was independent of SWI/SNF complexes activity as observed in C2C12 cells. 84 BRM was required for glucocorticoids to get access to the pro- The SWI/SNF-mediated chromatin remodelling mechanism has been proved to exist in cartilage tissue. 11 genes of SWI/SNF complexes including BRG1 and BRM were identified by comprehensive mapping of human cartilage-expressed transcripts. 89 During BMP2-induced chondrogenic differentiation of C3H10T1/2 cells, the expression of fibroblast growth factor receptor 3 (Fgfr3) was upregulated by transcription factor SP1. The regulatory mechanism of this process was revealed as BRG1 exposed SP1 binding sites at Fgfr3 promoter. 90

| ASH1L
Absent, small, or homeotic disc1 like (ASH1L) is another member of the Trithorax group containing a SET domain and is known as an epigenetic transcriptional activator counteracting Polycomb repression. 91 A recent study revealed the potential role of ASH1L in the epigenetic regulation of MSCs differentiation. Levels of ASH1L were reduced in mice osteoporosis models as well as human osteoporotic samples, indicating a positive correlation of Ash1l expression with bone mass.
Ash1l knockdown suppressed osteogenic and chondrogenic differentiation of C3H10T1/2 cells via decreasing H3K4me3 deposition on the promoter region of Hoxa10, Osx, Runx2, and Sox9 genes. These results hint the role of ASH1L in osteogenesis and chondrogenesis of MSCs is dependent on its HMTase activity. 92 Meanwhile, it has been demonstrated that ASH1L occupied transcriptional regions of abundant active genes including the HOX genes and was responsible for H3K4 trimethylation. 93 Another group of studies reported that mammalian ASH1L specifically mono-or di-methylated histone H3K36 based on experiments using nucleosomes as substrates. 94,95 The controversial observation of its HMTase activity raises the possibility that ASH1L may have an unrevealed role in transcription regulation and stem cell fate determination.

| CBP/p300
The Trx complex named TAC1 in Drosophila contains CBP which interacts with Ash1 and confronts Polycomb silencing. 96 The mammalian CBP and its paralogue p300, encoded by CREBBP/EP300 genes, exhibit HAT activity that is responsible for H3K27 acetylation (H3K27ac). 22 As important transcriptional co-activators, CBP and p300 regulate the expression of a broad range of genes, especially transcription factors, that are involved in cell proliferation, differentiation and other cellular processes.
Homozygous null mutations of CBP or p300 in mice cause early embryonic lethality, and Crebbp heterozygous mice show various tissue defects involving the skeletal, haematopoietic and nervous system. It has been found that heterozygous Crebbp-deficient mice showed a significant decrease in trabecular bone volume mainly related to osteoclastogenesis of bone marrow stromal cells since the mineral apposition or bone formation rates are unperturbed. 97 In humans, monoallelic mutations in either CREBBP or EP300 cause the Rubinstein-Taybi syndrome, which is autosomal dominant inheritance and exhibits congenital abnormalities such as distinctive facial features, skeletal dysplasia, and intellectual disability. 98,99 The mechanism of CBP/p300 in gene expression activation has been well established. CBP/p300 associates with gene enhancer and TSS regions, bind to various transcription factors bridging them to