👤 Marc H W A Wijnen

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis. Show less

Fluorescence-guided surgery (FGS), based on fluorescent tracers binding to tumor-specific biomarkers, could assist surgeons to achieve complete tumor resections. This study evaluated potential biomarkers for FGS in pediatric Ewing sarcoma (ES). Immunohistochemistry (IHC) was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. LINGO-1 and GD2 evaluation did not work on the acquired tissue. Based on the immunoreactive scores, anti-CD99 and anti-CD117 were evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. Anti-GD2, a tracer in the developmental phase, was also tested. These three tracers were topically applied to a freshly resected ES tumor and adjacent healthy tissue. IHC demonstrated moderate/strong CD99 and CD117 expression in ES tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117 and low GD2 expression, in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient. In conclusion, CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients. Show less

Epigenetic regulatory proteins support mammalian development, cancer, aging and tissue repair by controlling many cellular processes including stem cell self-renewal, lineage-commitment and senescence in both skeletal and non-skeletal tissues. We review here our knowledge of epigenetic regulatory protein complexes that support the formation of inaccessible heterochromatin and suppress expression of cell and tissue-type specific biomarkers during development. Maintenance and formation of heterochromatin critically depends on epigenetic regulators that recognize histone 3 lysine trimethylation at residues K9 and K27 (respectively, H3K9me3 and H3K27me3), which represent transcriptionally suppressive epigenetic marks. Three chromobox proteins (i.e., CBX1, CBX3 or CBX5) associated with the heterochromatin protein 1 (HP1) complex are methyl readers that interpret H3K9me3 marks which are mediated by H3K9 methyltransferases (i.e., SUV39H1 or SUV39H2). Other chromobox proteins (i.e., CBX2, CBX4, CBX6, CBX7 and CBX8) recognize H3K27me3, which is deposited by Polycomb Repressive Complex 2 (PRC2; a complex containing SUZ12, EED, RBAP46/48 and the methyl transferases EZH1 or EZH2). This second set of CBX proteins resides in PRC1, which has many subunits including other polycomb group factors (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, PCGF6), human polyhomeotic homologs (HPH1, HPH2, HPH3) and E3-ubiquitin ligases (RING1 or RING2). The latter enzymes catalyze the subsequent mono-ubiquitination of lysine 119 in H2A (H2AK119ub). We discuss biological, cellular and molecular functions of CBX proteins and their physiological and pathological activities in non-skeletal cells and tissues in anticipation of new discoveries on novel roles for CBX proteins in bone formation and skeletal development. Show less

Control of gene expression by epigenetic regulators is fundamental to tissue development and homeostasis. Loss-of-function (LOF) studies using siRNAs for epigenetic regulators require that RNA interference rapidly reduces the cellular levels of the corresponding mRNAs and/or proteins. The most abundant chromatin structural proteins (i.e., the core histones H2A, H2B, H3 and H4) have relatively long half-lives and do not turn over rapidly, although their mRNAs are labile. The question arises whether epigenetic regulatory enzymes (e.g., Ezh2) or proteins that interact with histones via selective modifications (e.g., Cbx1 to Cbx8, Brd4) are stable or unstable. Therefore, we performed classical α-amanitin and cycloheximide inhibition assays that block, respectively, mRNA transcription and protein translation in mouse MC3T3 osteoblasts, ATDC5 chondrocytes and C2C12 myoblasts. We find that mRNA levels of Cbx proteins and Ezh2 were significantly depleted after 24 hrs, while their corresponding proteins remained relatively stable. As positive control, the half-life of the labile cyclin D1 protein was found to be less than 1 hr. Our study suggests that histone code readers and writers are relatively stable chromatin-related proteins, which is consistent with their long-term activities in maintaining chromatin organization and phenotype identity. These findings have conceptual ramifications for the interpretation of RNAi experiments that reduce the mRNA but not protein levels of epiregulatory proteins. We propose that siRNAs for at least some epigenetic regulatory proteins may exert their biological effects by blocking translation and new protein synthesis rather than by decreasing pre-existing protein pools. Show less

Heparan sulfate proteoglycans (HSPGs) are key components of the extracellular matrix that mediate cell proliferation, invasion, and cellular signaling. The biological functions of HSPGs are linked to their co-stimulatory effects on extracellular ligands (e.g., WNTs) and the resulting activation of transcription factors that control mammalian development but also associated with tumorigenesis. We examined the expression profile of HSPG core protein syndecans (SDC1-4) and glypicans (GPC1-6) along with the enzymes that initiate or modify their glycosaminoglycan chains in human breast cancer (HBC) epithelial cells. Gene expression in relation to cell proliferation was examined in the HBC cell lines MCF-7 and MDA-MB-231 following treatment with the HS agonist heparin. Heparin increased gene expression of chain initiation and modification enzymes including EXT1 and NDST1, as well as core proteins SDC2 and GPC6. With HS/Wnt interactions established, we next investigated WNT pathway components and observed that increased proliferation of the more invasive MDA-MB-231 cells is associated with activation of the Wnt signaling pathway. Specifically, there was substantial upregulation (>5-fold) of AXIN1, WNT4A, and MYC in MDA-MB-231 but not in MCF-7 cells. The changes in gene expression observed for HSPG core proteins and related enzymes along with the associated Wnt signaling components suggest coordinated interactions. The influence of HSPGs on cellular proliferation and invasive potential of breast cancer epithelial cells are cell and niche specific. Further studies on the interactions between HSPGs and WNT ligands may yield clinically relevant molecular targets, as well as new biomarkers for characterization of breast cancer progression. Show less

In yeast, the G1 cyclin Cln3 promotes cell cycle entry by activating the transcription factor SBF. In mammals, there is a parallel system for cell cycle entry in which cyclin dependent kinase (CDK) activates transcription factor E2F/Dp. Here we show that Cln3 regulates SBF by at least two different pathways, one involving the repressive protein Whi5, and the second involving Stb1. The Rpd3 histone deacetylase complex is also involved. Cln3 binds to SBF at the CLN2 promoter, and removes previously bound Whi5 and histone deacetylase. Adding extra copies of the SBF binding site to the cell delays Start, possibly by titrating Cln3. Since Rpd3 is the yeast ortholog of mammalian HDAC1, there is now a virtually complete analogy between the proteins regulating cell cycle entry in yeast (SBF, Cln3, Whi5 and Stb1, Rpd3) and mammals (E2F, Cyclin D, Rb, HDAC1). The cell may titrate Cln3 molecules against the number of SBF binding sites, and this could be the underlying basis of the size-control mechanism for Start. Show less

Heparan sulfate proteoglycans cooperate with basic fibroblast growth factor (bFGF/FGF2) signaling to control osteoblast growth and differentiation, as well as metabolic functions of osteoblasts. FGF2 signaling modulates the expression and activity of Runt-related transcription factor 2 (Runx2/Cbfa1), a key regulator of osteoblast proliferation and maturation. Here, we have characterized novel Runx2 target genes in osteoprogenitors under conditions that promote growth arrest while not yet permitting sustained phenotypic maturation. Runx2 enhances expression of genes related to proteoglycan-mediated signaling, including FGF receptors (e.g., FGFR2 and FGFR3) and proteoglycans (e.g., syndecans [Sdc1, Sdc2, Sdc3], glypicans [Gpc1], versican [Vcan]). Runx2 increases expression of the glycosyltransferase Exostosin-1 (Ext1) and heparanase, as well as alters the relative expression of N-linked sulfotransferases (Ndst1 = Ndst2 > Ndst3) and enzymes mediating O-linked sulfation of heparan sulfate (Hs2st > Hs6st) or chondroitin sulfate (Cs4st > Cs6st). Runx2 cooperates with FGF2 to induce expression of Sdc4 and the sulfatase Galns, but Runx2 and FGF2 suppress Gpc6, thus suggesting intricate Runx2 and FGF2 dependent changes in proteoglycan utilization. One functional consequence of Runx2 mediated modulations in proteoglycan-related gene expression is a change in the responsiveness of bone markers to FGF2 stimulation. Runx2 and FGF2 synergistically enhance osteopontin expression (>100 fold), while FGF2 blocks Runx2 induction of alkaline phosphatase. Our data suggest that Runx2 and the FGF/proteoglycan axis may form an extracellular matrix (ECM)-related regulatory feed-back loop that controls osteoblast proliferation and execution of the osteogenic program. Show less

In Saccharomyces cerevisiae (budding yeast), commitment to cell division in late G(1) is promoted by the G(1) cyclin Cln3 and its associated cyclin-dependent kinase, Cdc28. We show here that all known aspects of the function of Cln3 in G(1) phase, including control of cell size, pheromone sensitivity, cell cycle progress, and transcription, require the protein Swi6. Swi6 is a component of two related transcription factors, SBF and MBF, which are known to regulate many genes at the G(1)-S transition. The Cln3-Cdc28 complex somehow activates SBF and MBF, but there was no evidence for direct phosphorylation of SBF/MBF by Cln3-Cdc28 or for a stable complex between SBF/MBF and Cln3-Cdc28. The activation also does not depend on the ability of Cln3 to activate transcription when artificially recruited directly to a promoter. The amino terminus and the leucine zipper of Swi6 are important for the ability of Swi6 to respond to Cln3 but are not essential for the basal transcriptional activity of Swi6. Cln3-Cdc28 may activate SBF and MBF indirectly, perhaps by phosphorylating some intermediary protein. Show less

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription. Show less

The transcription complexes SBF and MBF mediate the G(1)-S transition in the cell cycle of Saccharomyces cerevisiae. In late G(1), SBF and MBF induce a burst of transcription in a number of genes, including G(1)- and S-phase cyclins. Activation of SBF and MBF depends on the G(1) cyclin Cln3 and a largely uncharacterized protein called Bck2. We show here that the induction of SBF/MBF target genes by Bck2 depends partly, but not wholly, on SBF and MBF. Unlike Cln3, Bck2 is capable of inducing its transcriptional targets in the absence of functional Cdc28. Our results revealed promoter-specific mechanisms of regulation by Cln3, Bck2, SBF, and MBF. We isolated high-copy suppressors of the cln3 bck2 growth defect; all of these had the ability to increase CLN2 expression. One of these suppressors was the negative regulator of meiosis RME1. Rme1 induces CLN2, and we show that it has a haploid-specific role in regulating cell size and pheromone sensitivity. Genetic analysis of the cln3 bck2 defect showed that CLN1, CLN2, and other SBF/MBF target genes have an essential role in addition to the degradation of Sic1. Show less