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Purpose of ReviewAlthough many signalling pathways have been discovered to be essential in mesenchymal stem/stromal (MSC) differentiation, it has become increasingly clear in recent years that epigenetic regulation of gene transcription is a vital component of lineage determination, encompassing diet, lifestyle and parental influences on bone, fat and cartilage development.Recent FindingsThis review discusses how specific enzymes that modify histone methylation and acetylation or DNA methylation orchestrate the differentiation programs in lineage determination of MSC and the epigenetic changes that facilitate development of bone related diseases such as osteoporosis. The review also describes how environmental factors such as mechanical loading influence the epigenetic signatures of MSC, and how the use of chemical agents or small peptides can regulate epigenetic drift in MSC populations during ageing and disease.SummaryEpigenetic regulation of MSC lineage commitment is controlled through changes in enzyme activity, which modifies DNA and histone residues leading to alterations in chromatin structure. The co-ordinated epigenetic regulation of transcriptional activation and repression act to mediate skeletal tissue homeostasis, where deregulation of this process can lead to bone loss during ageing or osteoporosis.

Background The 5 hydroxymethylation (5hmC) mark and TET DNA dioxygenases play a pivotal role in embryonic stem cell differentiation and animal development. However, very little is known about TET enzymes in lineage determination of human bone marrow-derived mesenchymal stem/stromal cells (BMSC). We examined the function of all three TET DNA dioxygenases, responsible for DNA hydroxymethylation, in human BMSC cell osteogenic and adipogenic differentiation. Results We used siRNA knockdown and retroviral mediated enforced expression of TET molecules and discovered TET1 to be a repressor of both osteogenesis and adipogenesis. TET1 was found to recruit the co-repressor proteins, SIN3A and the histone lysine methyltransferase, EZH2 to osteogenic genes. Conversely, TET2 was found to be a promoter of both osteogenesis and adipogenesis. The data showed that TET2 was directly responsible for 5hmC levels on osteogenic and adipogenic lineage-associated genes, whereas TET1 also played a role in this process. Interestingly, TET3 showed no functional effect in BMSC osteo-/adipogenic differentiation. Finally, in a mouse model of ovariectomy-induced osteoporosis, the numbers of clonogenic BMSC were dramatically diminished corresponding to lower trabecular bone volume and reduced levels of TET1, TET2 and 5hmC. Conclusion The present study has discovered an epigenetic mechanism mediated through changes in DNA hydroxymethylation status regulating the activation of key genes involved in the lineage determination of skeletal stem cells, which may have implications in BMSC function during normal bone regulation. Targeting TET molecules or their downstream targets may offer new therapeutic strategies to help prevent bone loss and repair following trauma or disease. Electronic supplementary material The online version of this article (10.1186/s13072-018-0247-4) contains supplementary material, which is available to authorized users.

There is mounting evidence in the literature that mesenchymal stromal/stem cell (MSC) like populations derived from different tissues, undergo epigenetic changes during aging, leading to compromised connective tissue integrity and function. This body of work has linked the biological aging of MSC to changes in their epigenetic signatures affecting growth, lifespan, self-renewal and multi-potential, due to deregulation of processes such as cellular senescence, oxidative stress, DNA damage, telomere shortening and DNA damage. This review addresses recent findings examining DNA methylation, histone modifications and miRNA changes in aging MSC populations. Moreover, we explore how epigenetic factors alter cellular pathways and associated biological networks, contributing to the MSC aging phenotype. Finally we discuss the crucial areas requiring a greater understanding of these processes, in order to piece together a global picture of the changing epigenetic landscape in MSC during aging.

In this study, we examined the functional importance of EZH2 during skeletal development and homeostasis using the conditional deletion of Ezh2 (Ezh2fl/fl ) in early mesenchyme with the use of a Prrx-1-cre driver mouse (Ezh2+/+). Heterozygous (Ezh2+/-) newborn and 4-wk-old mice exhibited increased skeletal size, growth plate size, and weight when compared to the wild-type control (Ezh2+/+), whereas homozygous deletion of Ezh2 (Ezh2-/-) resulted in skeletal deformities and reduced skeletal size, growth plate size, and weight in newborn and 4-wk-old mice. Ezh2-/- mice exhibited enhanced trabecular patterning. Osteogenic cortical and trabecular bone formation was enhanced in Ezh2+/- and Ezh2-/- animals. Ezh2+/- and Ezh2-/- mice displayed thinner cortical bone and decreased mechanical strength compared to the wild-type control. Differences in cortical bone thickness were attributed to an increased number of osteoclasts, corresponding with elevated levels of the bone turnover markers cross-linked C-telopeptide-1 and tartrate-resistant acid phosphatase, detected within serum. Moreover, Ezh2+/- mice displayed increased osteoclastogenic potential coinciding with an upregulation of Rankl and M-csf expression by mesenchymal stem cells (MSCs). MSCs isolated from Ezh2+/- mice also exhibited increased trilineage potential compared with wild-type bone marrow stromal/stem cells (BMSCs). Gene expression studies confirmed the upregulation of known Ezh2 target genes in Ezh2-/- bone tissue, many of which are involved in Wnt/BMP signaling as promoters of osteogenesis and inhibitors of adipogenesis. In summary, EZH2 appears to be an important orchestrator of skeletal development, postnatal bone remodelling and BMSC fate determination in vitro and in vivo-Hemming, S., Cakouros, D., Codrington, J., Vandyke, K., Arthur, A., Zannettino, A., Gronthos, S. EZH2 deletion in early mesenchyme compromises postnatal bone microarchitecture and structural integrity and accelerates remodeling.

The methyltransferase, Enhancer of Zeste homology 2 (EZH2), trimethylates histone 3 lysine 27 (H3K27me3) on chromatin and this repressive mark is removed by lysine demethylase 6A (KDM6A). Loss of these epigenetic modifiers results in developmental defects. We demonstrate that Ezh2 and Kdm6a transcript levels change during differentiation of multipotential human bone marrow-derived mesenchymal stem cells (MSC). Enforced expression of Ezh2 in MSC promoted adipogenic in vitro and inhibited osteogenic differentiation potential in vitro and in vivo, whereas Kdm6a inhibited adipogenesis in vitro and promoted osteogenic differentiation in vitro and in vivo. Inhibition of EZH2 activity and knockdown of Ezh2 gene expression in human MSC resulted in decreased adipogenesis and increased osteogenesis. Conversely, knockdown of Kdm6a gene expression in MSC leads to increased adipogenesis and decreased osteogenesis. Both Ezh2 and Kdm6a were shown to affect expression of master regulatory genes involved in adipogenesis and osteogenesis and H3K27me3 on the promoters of master regulatory genes. These findings demonstrate an important epigenetic switch centered on H3K27me3 which dictates MSC lineage determination. Stem Cells 2014;32:802–815

Histone three lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) is a critical epigenetic modifier, which regulates gene transcription through the trimethylation of the H3K27 residue leading to chromatin compaction and gene repression. EZH2 has previously been identified to regulate human bone marrow-derived mesenchymal stem cells (MSC) lineage specification. MSC lineage specification is regulated by the presence of EZH2 and its H3K27me3 modification or the removal of the H3K27 modification by lysine demethylases 6A and 6B (KDM6A and KDM6B). This study used a bioinformatics approach to identify novel genes regulated by EZH2 during MSC osteogenic differentiation. In this study, we identified the EZH2 targets, ZBTB16, MX1, and FHL1, which were expressed at low levels in MSC. EZH2 and H3K27me3 were found to be present along the transcription start site of their respective promoters. During osteogenesis, these genes become actively expressed coinciding with the disappearance of EZH2 and H3K27me3 on the transcription start site of these genes and the enrichment of the active H3K4me3 modification. Overexpression of EZH2 downregulated the transcript levels of ZBTB16, MX1, and FHL1 during osteogenesis. Small interfering RNA targeting of MX1 and FHL1 was associated with a downregulation of the key osteogenic transcription factor, RUNX2, and its downstream targets osteopontin and osteocalcin. These findings highlight that EZH2 not only acts through the direct regulation of signaling modules and lineage-specific transcription factors but also targets many novel genes important for mediating MSC osteogenic differentiation.

Twist-1 encodes a basic helix-loop-helix transcription factor, known to contribute to mesodermal and skeletal tissue development. We have reported previously that Twist-1 maintains multipotent human bone marrow-derived mesenchymal stem/stromal cells (BMSC) in an immature state, enhances their life-span, and influences cell fate determination. In this study, human BMSC engineered to express high levels of Twist-1 were found to express elevated levels of the chemokine, CXCL12. Analysis of the CXCL12 proximal promoter using chromatin immunoprecipitation analysis identified several E-box DNA sites bound by Twist-1. Functional studies using a luciferase reporter construct showed that Twist-1 increased CXCL12 promoter activity in a dose dependent manner. Notably, Twist-1 over-expressing BMSC exhibited an enhanced capacity to maintain human CD34 + hematopoietic stem cells (HSC) in long-term culture-initiating cell (LTC-IC) assays. Moreover, the observed increase in HSC maintenance by Twist-1 over-expressing BMSC was blocked in the presence of the CXCL12 inhibitor, AMD3100. Supportive studies, using Twist-1 deficient heterozygous mice demonstrated a significant decrease in the frequency of stromal progenitors and increased numbers of osteoblasts within the bone. These observations correlated to a decreased incidence in the number of clonogenic stromal progenitors (colony forming unit–fibroblasts) and lower levels of CXCL12 in Twist-1 mutant mice. Furthermore, Twist-1 deficient murine stromal feeder layers, exhibited a significant decrease in CXCL12 levels and lower numbers of hematopoietic colonies in LTC-IC assays, compared with wild type controls. These findings demonstrate that Twist-1, which maintains BMSC at an immature state, endows them with an increased capacity for supporting hematopoiesis via direct activation of CXCL12 gene expression.

In this review we explore the importance of epigenetics as a contributing factor for aging adult stem cells. We summarize the latest findings of epigenetic factors deregulated as adult stem cells age and the consequence on stem cell self-renewal and differentiation, with a focus on adult stem cells in the bone marrow. With the latest whole genome bisulphite sequencing and chromatin immunoprecipitations we are able to decipher an emerging pattern common for adult stem cells in the bone marrow niche and how this might correlate to epigenetic enzymes deregulated during aging. We begin by briefly discussing the initial observations in yeast, drosophila and Caenorhabditis elegans (C. elegans) that led to the breakthrough research that identified the role of epigenetic changes associated with lifespan and aging. We then focus on adult stem cells, specifically in the bone marrow, which lends strong support for the deregulation of DNA methyltransferases, histone deacetylases, acetylates, methyltransferases and demethylases in aging stem cells, and how their corresponding epigenetic modifications influence gene expression and the aging phenotype. Given the reversible nature of epigenetic modifications we envisage “epi” targeted therapy as a means to reprogram aged stem cells into their younger counterparts.

The main impairment to tissue maintenance during aging is the reduced capacity for stem cell self-renewal over time due to senescence, the irreversible block in proliferation. We have previously described that the basic helix-loop-helix (bHLH) transcription factor Twist-1 can greatly enhance the life span of bone marrow-derived mesenchymal stem/stromal cells (MSCs). In the present study, we show that Twist-1 potently suppresses senescence and the Ink4A/Arf locus with a dramatic decrease in the expression of p16 and to some extent a decrease in p14. Furthermore, the polycomb group protein and histone methyltransferase Ezh2, which suppresses the Ink4A/Arf locus, was found to be induced by Twist-1, resulting in an increase in H3K27me3 along the Ink4A/Arf locus, repressing transcription of both p16/p14 and senescence of human MSCs. Furthermore, Twist-1 inhibits the expression of the bHLH transcription factor E47, which is normally expressed in senescent MSCs and induces transcription of the p16 promoter. Reduced Twist-1 wild-type expression and function in bone cells derived from Saethre-Chotzen patients also revealed an increase in senescence. These studies for the first time link Twist-1 to histone methylation of the Ink4A/Arf locus by controlling the expression of histone methyltransferases as well as the expression of other bHLH factors.

Bone marrow-derived mesenchymal stem cells (MSC), are multipotent cells that give rise to multiple lineages including osteoblasts, adipocytes, muscle, and fibroblasts. MSCs are useful for clinical applications such as cell therapy because they can be isolated from an individual and expanded for use in tissue repair, as well as other therapeutic applications, without immune rejection. However, one of the key problems in the use of MSCs for these applications is the efficiency of these cells to engraft and fully regenerate damaged tissues. Therefore, to optimize this process, a comprehensive understanding of the key regulators of MSCs self-renewal and maintenance are critical to the success of future cell therapy as well as other clinical applications. The basic helix loop helix transcription factor, Twist, plays a master regulatory role in all of these processes and, therefore, a thorough understanding of the mechanistic insights in the role of Twist in lineage specification/differentiation and tumorigenesis is vital to the success of future clinical applications for the therapeutic use of MSCs. In this article, we highlight the basic mechanisms and signaling pathways that are important to MSC fate, maintenance, and differentiation, as well as the critical role that Twist plays in these processes. In addition, we review the known literature suggesting a critical role for Twist in the generation of cancer stem cells, as this information may contribute to a broader understanding of stem cell biology and stem-cell-based therapeutics.

Human BMSSCs lose telomerase activity in vitro, which leads to chromosomal instability and cellular senescence. We observed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. We showed that Cbfa1 acts as a partial repressor of TERT, which may facilitate cellular differentiation. Introduction: The absence of telomerase activity by cultured human bone marrow stromal stem cells (BMSSCs) causes critical shortening of chromosomal telomeres, leading eventually to cellular senescence. Ex vivo expansion of BMSSCs correlates to an increase in osteogenic lineage associated markers such as alkaline phosphatase, bone sialoprotein, and osteocalcin that are regulated by the master regulatory transcription factor, Cbfa1 (Runx2). This study examined whether Cbfa1 was capable of regulating the promoter of the early stem cell-associated gene, telomerase reverse transcriptase (TERT). Materials and Methods: Human BMSSCs were isolated by fluorescence-activated cell sorting. Telomerase activity was determined using the telometric repeat amplification protocol. CBFA1 and TERT gene expression was assessed by real-time PCR. The functional capacity of Cbfa1 to bind to the hTERT promoter was performed using a modified electrophoretic mobility shift assay (EMSA). Chromatin immunoprecipitation (ChIP) analysis was used to examine Cbfa1 binding to the hTERT promoter in vivo. Functional analysis of CBFA-1 wildtype and mutant DNA binding sites on TERT promoter fragments was assessed using the promoterless green fluorescence protein (GFP) reporter vector, pEGFP-1, after transfection into HOS cells. Results: This study showed an inverse expression pattern between the osteogenic master regulatory gene, CBFA1, and the stem cell-associated gene, hTERT. The data showed that BMSSCs undergo osteogenic commitment after the loss of hTERT expression, with concomitant elevated levels of CBFA1 transcripts. In addition, two unique Cbfa1 DNA binding sites were identified on the hTERT proximal promoter by EMSA supershift assay. Mutated forms of the putative Cbfa1 binding sites, created by site-directed mutagenesis, were able to abolish this interaction. ChIP analysis showed that Cbfa1 interacted directly with the hTERT promoter in vivo. Functional studies using GFP reporter constructs, driven by 2- and 3-kbp hTERT proximal promoter fragments, showed significantly lower levels of transcriptional activity compared with corresponding constructs with mutated Cbfa1 binding site Oligo 2. Conclusions: These studies suggest that Cbfa1 may act as a repressor of early stem cell markers such as hTERT as one possible mechanism for facilitating cellular differentiation. J Bone Miner Res 2007;22:897-906. Published online on March 12, 2007; doi: 10.1359/JBMR.070308

Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. Here we show that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. We demonstrate that dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. We further show that salivary gland degradation requires dUTX catalytic activity. Our findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development. Refereed/Peer-reviewed

Basic helix-loop-helix (bHLH) transcription factors are pivotal regulators of cellular differentiation and development. The bHLH factor, Twist-1 has previously been found to control bone marrow stromal/stem cells (BMSC) self-renewal, life span, and differentiation, however not much is known about its mechanism of action. In this study, we have discovered a novel Twist-1 regulated bHLH gene, Hes4, expressed in humans, but not in mice. Its closest homologue in both humans and mice is Hes1. Overexpression and knockdown studies demonstrated that Hes4 promotes osteogenesis resulting in an increase in Runx2, osteocalcin, osteopontin, and bone sialoprotein expression. Conversely, Hes4 was found to inhibit adipogenesis accompanied by a decrease in PPARγ2, adiponectin, and adipsin expression. In vitro studies indicate that Hes4 employs a mechanism to counteract the negative function of Twist-1 on osteogenesis by binding to Twist-1 and inhibiting the ability of Twist-1 to bind and inhibit Runx2. In vivo chromatin immunoprecipitation and in vitro reporter assays illustrated that Runx2 recruitment to the osterix promoter, was found to be enhanced in the presence of Hes4 and inhibited in the presence of Twist-1. Therefore, Hes4 antagonizes the function of Twist-1 to regulate lineage commitment of BMSC. These studies highlight the potential differences in molecular mechanisms that regulate BMSC osteogenic differentiation between human and mouse.

The steroid hormone ecdysone regulates moulting, cell death, and differentiation during insect development. Ecdysone mediates its biological effects by either direct activation of gene transcription after binding to its receptor EcR–Usp or via hierarchical transcriptional regulation of several primary transcription factors. In turn, these transcription factors regulate the expression of several downstream genes responsible for specific biological outcomes. DRONC, the Drosophila initiator caspase, is transcriptionally regulated by ecdysone during development. We demonstrate here that the dronc promoter directly binds EcR–Usp. We further show that mutation of the EcR–Usp binding element (EcRBE) reduces transcription of a reporter and abolishes transactivation by an EcR isoform. We demonstrate that EcRBE is required for temporal regulation of dronc expression in response to ecdysone in specific tissues. We also uncover the participation of a putative repressor whose function appears to be coupled with EcR–Usp. These results indicate that direct binding of EcR–Usp is crucial for controlling the timing of dronc expression in specific tissues.

Developmental and physiological cues, as well as signals in response to cell damage, initiate a cell suicide program that is often referred to as apoptosis or programmed cell death (PCD). PCD is mediated by the core cell-death machinery consisting of proteins that mediate and control the activation of caspases – enzymes that dismantle cellular architecture during apoptosis. Because components of the caspase-activation apparatus are constitutively present in most cells, most studies during the last decade have focused on the posttranslational regulation of this preexisting machinery. However, accumulating evidence suggests that, in many contexts, transcriptional regulation also plays a crucial role in the activation of the physiological cell-death program.

Developmentally programmed cell death is regulated by a balance between pro- and anti-death signaling. During Drosophila metamorphosis, the removal of larval tissues is dependent on the steroid hormone ecdys one, which controls the levels of pro- and anti-death molecules. Ecdysone binds to its heterodimeric receptor ecdysone receptor/ultraspiracle to mediate transcription of primary response genes. Here we show that CARMER, an arginine-histone methyltransferase, is critical in coordinating ecdysone-induced expression of Drosophila cell death genes. Ablation of CARMER blocks ecdysone-induced cell death in Drosophila cells, but not apoptosis induced by cell stress. We demonstrate that CARMER associates with the ecdysone receptor complex and modulates the ecdysone-induced transcription of a number of apoptotic genes. Thus, the chromatin-modifying protein, CARMER, modulates cell death by controlling the hormone-dependent expression of the core cell death machinery.

The steroid hormone ecdysone regulates both cell differentiation and cell death during insect metamorphosis, by hierarchical transcriptional regulation of a number of genes, including the Broad-Complex (BR-C), the zinc finger family of transcription factors. These genes in turn regulate the transcription of a number of downstream genes. DRONC, a key apical caspase in Drosophila, is the only known caspase that is transcriptionally regulated by ecdysone during development. We demonstrate that dronc gene expression is ablated or reduced in BR-C mutant flies. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that DRONC is essential for ecdysone-mediated cell death, and that dronc upregulation in these cells is controlled by BR-C. Finally, we show that the dronc promoter has BR-C interaction sites, and that it can be transactivated by a specific isoform of BR-C. These results indicate that BR-C plays a key role in ecdysone-mediated caspase regulation.

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-2 −/− mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with E1A and Ras oncogenes, caspase-2 −/− MEFs grew significantly faster than caspase-2 +/+ MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse Eμ- Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-2 −/− mice. The caspase-2 −/− cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-2 −/− MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following γ-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein.

Online supplemental material: Fig. S1 shows localization of dLKR/SDH in transfected cells. Fig. S2 shows quantifi cation of the RNAi-mediated knockdown of dLKR/SDH. Online supplemental material is available at http://www.jcb.org/cgi/content/ full/jcb.200712169/DC1. The sequential modifi cations of histones form the basis of the histone code that translates into either gene activation or repression. Nuclear receptors recruit a cohort of histone-modifying enzymes in response to ligand binding and regulate proliferation, differentiation, and cell death. In Drosophila melanogaster, the steroid hormone ecdysone binds its heterodimeric receptor ecdysone receptor/ultraspiracle to spatiotemporally regulate the transcription of several genes. In this study, we identify a novel cofactor, Drosophila lysine ketoglutarate reductase (dLKR)/saccharopine dehydrogenase (SDH), that is involved in ecdysone-mediated transcription. dLKR/SDH binds histones H3 and H4 and suppresses ecdysonemediated transcription of cell death genes by inhibiting histone H3R17me2 mediated by the Drosophila arginine methyl transferase CARMER. Our data suggest that the dynamic recruitment of dLKR/SDH to ecdysone-regulated gene promoters controls the timing of hormone-induced gene expression. In the absence of dLKR/SDH, histone methylation occurs prematurely, resulting in enhanced gene activation. Consistent with these observations, the loss of dLKR/SDH in Drosophila enhances hormone-regulated gene expression, affecting the developmental timing of gene activation.

Hui and colleagues have raised the concern that the detection of cell surface Annexin V staining on mesenchymal stem cells (MSC) following treatment with the enhancer of Zeste homology 2 (EZH2) inhibitor, DZNep (Sequoia Research, Pangbourne, United States, http:// www.seqchem.com/) may have been compromised due to the use of Trypsin/EDTA to detach the adherent cells in our recent publication “EZH2 and KDM6A Act as an Epigenetic Switch to Regulate MSC lineage specification” [1]. Specifically, it is proposed that EDTA could affect the structure of the plasma membrane and chelate Ca preventing the binding of Annexin V protein to the phosphatidylserine [2–4]. However, different manufacturers specify the use of Trypsin/EDTA, and not EDTA-free trypsin for detaching adherent cells prior to staining for Annexin V, where Trypsin/EDTA has been used by various studies for a wide range of adherent cell types [5–12]. While the Letter to the Editor by Hui and colleagues, published in this article, use the example that DZNep has been previously shown to induced apoptosis in human colon cancer stem cells [13], our study [1] found that DZNep had no effect on the apoptosis status of human bone marrow-derived MSC, in accord with other studies reporting that DZNeP is not harmful for nontransformed cells at tumor-inhibiting doses [14]. The discrepancy in DZNep sensitivity between normal stem cells and cancer stem cells may be due to aberrant levels of EZH2 in cancer cells required for survival [15, 16]. In order to determine whether EDTA influences Annexin V binding on MSC, we performed the Annexin V protocol with either Trypsin/EDTA (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) as described in our published study [1], or with EDTA-free Collagenase I (2 mg/ml Worthington Biochemical Corporation, NJ, United States of America, http://www.worthington-biochem. com/default.html). Two different human MSC cultures were treated with 5 mM of the potent apoptotic drug, doxorubicin (Sapphire Biosciences, NSW, Australia, http://www.sapphirebiosci ence.com/) for 24 hours prior to Annexin V staining. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then washed with Hanks Balanced Salt Solution (Sigma Aldrich, NSW, Australia, http://www.sigmaaldrich.com/australia.html) supplemented with 5% fetal calf serum (FCS; SAFC Biosciences, Sydney, NSW, Australia, http://www.safcglobal.com/safc-global/en-us/ home.html). The cells were further washed twice with cold phosphate buffered saline prior to cell staining for Annexin V as specified in the manufacturer’s protocol (Annexin V conjugates Alexa Fluro 488, for apoptosis detection, Life Technologies, VIC, Australia, http:// www.lifetechnologies.com/au/). Flow cytometry was used to determine the levels of Annexin V staining under each condition. Cells treated with doxorubicin without Annexin V Figure 1. Comparable expression of Annexin V on doxorubicin treated MSC detached by Trypsin/EDTA or Collagenase I. Two different human bone marrow-derived MSC cultures were treated with 5 mM doxorubicin for 24 hours. Single-cell suspensions were harvested by either Trypsin/EDTA or Collagenase I treatment then stained for Annexin V. Assessment of Annexin V levels was performed by flow cytometric analysis. Data represent mean fluorescence intensity from three replicate experiments. Abbreviation: MSC, mesenchymal stem cells. Mesechymal Stem Cell Laboratory, School of Health Sciences, Faculty of Health Sciences and Centre for Stem Cell Research, University of Adelaide, South Australia, Australia

The Drosophila steroid hormone ecdysone mediates cell death during metamorphosis by regulating the transcription of a number of cell death genes. The apical caspase DRONC is known to be transcriptionally regulated by ecdysone during development. Here we demonstrate that ecdysone also regulates the transcription of DRICE, a major effector caspase and a downstream target for DRONC in the fly. Using RNA interference in an ecdysone-responsive Drosophila cell line, we show that drice up-regulation is essential for apoptosis induced by ecdysone. We also show that drice expression is specifically controlled by the ecdysone-regulated transcription factor BR-C. Combined with previous observations, our results indicate that transcriptional regulation of the components of the core apoptotic machinery plays a key role in hormone-regulated programmed cell death during Drosophila development.

The GM-CSF gene is expressed following activation of T cells. The proximal promoter and an upstream enhancer have previously been characterized using transfection and reporter assays in T cell lines in culture. A 10.5-kb transgene containing the entire human GM-CSF gene has also been shown to display inducible, position-independent, copy number-dependent transcription in mouse splenocytes. To determine the role of individual promoter elements in transgene function, mutations were introduced into the proximal promoter and activity assessed following the generation of transgenic mice. Of four mutations introduced into the transgene promoter, only one, in an NF-κB/Sp1 region, led to decreased induction of the transgene in splenocytes or bone marrow-derived macrophages. This mutation also affected the activity of reporter gene constructs stably transfected into T cell lines in culture, but not when transiently transfected into the same cell lines. The mutation alters the NF-κB family members that bind to the NF-κB site as well as reducing the binding of Sp1 to an adjacent element. A DNase I hypersensitive site that is normally generated at the promoter following T cell activation on the wild-type transgene does not appear in the mutant transgene. These results suggest that the NF-κB/Sp1 region plays a critical role in chromatin remodeling and transcription on the GM-CSF promoter in primary T cells.

The granulocyte macrophage colony stimulating factor (GM-CSF) promoter contains a 10 bp element known as CK-1 or CD28RE that specifically responds to the co-stimulatory signal delivered to T cells via the CD28 surface receptor. This element is a variant NFkappaB site that does not function alone but requires an adjacent promoter region that includes a classical NFkappaB element, an Sp-1 site and a putative activator protein-1 (AP-1)-like binding site. The entire region is referred to as the CD28 response region (CD28RR). The GM-CSF CK-1 element has been shown to bind NFkappaB proteins, in particular c-Rel, whose binding and function is dependent on the architectural transcription factor HMGI(Y). It has been previously suggested that the nuclear factor of activated T cells (NFAT) family of proteins also plays a role in the activity of this region. We show here that recombinant NFATp but not AP-1 can bind to the GM-CSF CD28RR. NFATp present in activated Jurkat T cell extracts can also interact with the CD28RR. The binding of NFATp and Rel proteins requires the same core CK-1 sequences, and appears to be mutually exclusive. We investigated the functional significance of NFATp binding to CK-1 by overexpressing the protein in Jurkat T cells and found that NFATp cannot activate the CD28RR alone but can cooperate with signals generated by phorbol 12-myristate 13-acetate/calcium ionophore. The CD28RR is therefore a complex region that can bind and respond to a combination of transcription factors and signals.