Non-canonical PRC1.1 Targets Active Genes Independent of H3K27me3 and Is Essential for Leukemogenesis.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

MicroRNA miR-378 promotes BMP2-induced osteogenic differentiation of mesenchymal progenitor cells.

BACKGROUND: MicroRNAs (miRNAs) are a family of small, non-coding single-stranded RNA molecules involved in post-transcriptional regulation of gene expression. As such, they are believed to play a role in regulating the step-wise changes in gene expression patterns that occur during cell fate specification of multipotent stem cells. Here, we have studied whether terminal differentiation of C2C12 myoblasts is indeed controlled by lineage-specific changes in miRNA expression. RESULTS: Using a previously generated RNA polymerase II (Pol-II) ChIP-on-chip dataset, we show differential Pol-II occupancy at the promoter regions of six miRNAs during C2C12 myogenic versus BMP2-induced osteogenic differentiation. Overexpression of one of these miRNAs, miR-378, enhances Alp activity, calcium deposition and mRNA expression of osteogenic marker genes in the presence of BMP2. CONCLUSIONS: Our results demonstrate a previously unknown role for miR-378 in promoting BMP2-induced osteogenic differentiation.

Dynamic modelling of microRNA regulation during mesenchymal stem cell differentiation.

BACKGROUND: Network inference from gene expression data is a typical approach to reconstruct gene regulatory networks. During chondrogenic differentiation of human mesenchymal stem cells (hMSCs), a complex transcriptional network is active and regulates the temporal differentiation progress. As modulators of transcriptional regulation, microRNAs (miRNAs) play a critical role in stem cell differentiation. Integrated network inference aimes at determining interrelations between miRNAs and mRNAs on the basis of expression data as well as miRNA target predictions. We applied the NetGenerator tool in order to infer an integrated gene regulatory network. RESULTS: Time series experiments were performed to measure mRNA and miRNA abundances of TGF-beta1+BMP2 stimulated hMSCs. Network nodes were identified by analysing temporal expression changes, miRNA target gene predictions, time series correlation and literature knowledge. Network inference was performed using NetGenerator to reconstruct a dynamical regulatory model based on the measured data and prior knowledge. The resulting model is robust against noise and shows an optimal trade-off between fitting precision and inclusion of prior knowledge. It predicts the influence of miRNAs on the expression of chondrogenic marker genes and therefore proposes novel regulatory relations in differentiation control. By analysing the inferred network, we identified a previously unknown regulatory effect of miR-524-5p on the expression of the transcription factor SOX9 and the chondrogenic marker genes COL2A1, ACAN and COL10A1. CONCLUSIONS: Genome-wide exploration of miRNA-mRNA regulatory relationships is a reasonable approach to identify miRNAs which have so far not been associated with the investigated differentiation process. The NetGenerator tool is able to identify valid gene regulatory networks on the basis of miRNA and mRNA time series data.

Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs.

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function.

The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation.

The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.

Mechanisms underlying the dualistic mode of action of major soy isoflavones in relation to cell proliferation and cancer risks.

Isoflavones are phytoestrogens that have been linked to both beneficial as well as adverse effects in relation to cell proliferation and cancer risks. The present article presents an overview of these seemingly contradicting health effects and of mechanisms that could be involved in this dualistic mode of action. One mechanism relates to the different ultimate cellular effects of activation of estrogen receptor (ER) α, promoting cell proliferation, and of ERß, promoting apoptosis, with the major soy isoflavones genistein and daidzein activating especially ERß. A second mode of action includes the role of epigenetics, including effects of isoflavones on DNA methylation, histone modification and miRNA expression patterns. The overview presented reveals that we are only at the start of unraveling the complex underlying mode of action for effects of isoflavones, both beneficial or adverse, on cell proliferation and cancer risks. It is evident that whatever model system will be applied, its relevance to human tissues with respect to ERα and ERß levels, co-repressor and co-activator characteristics as well as its relevance to human exposure regimens, needs to be considered and defined.

Epigenetics: DNA demethylation promotes skeletal myotube maturation.

Mesenchymal progenitor cells can be differentiated in vitro into myotubes that exhibit many characteristic features of primary mammalian skeletal muscle fibers. However, in general, they do not show the functional excitation-contraction coupling or the striated sarcomere arrangement typical of mature myofibers. Epigenetic modifications have been shown to play a key role in regulating the progressional changes in transcription necessary for muscle differentiation. In this study, we demonstrate that treatment of murine C2C12 mesenchymal progenitor cells with 10 µM of the DNA methylation inhibitor 5-azacytidine (5AC) promotes myogenesis, resulting in myotubes with enhanced maturity as compared to untreated myotubes. Specifically, 5AC treatment resulted in the up-regulation of muscle genes at the myoblast stage, while at later stages nearly 50% of the 5AC-treated myotubes displayed a mature, well-defined sarcomere organization, as well as spontaneous contractions that coincided with action potentials and intracellular calcium transients. Both the percentage of striated myotubes and their contractile activity could be inhibited by 20 nM TTX, 10 µM ryanodine, and 100 µM nifedipine, suggesting that action potential-induced calcium transients are responsible for these characteristics. Our data suggest that genomic demethylation induced by 5AC overcomes an epigenetic barrier that prevents untreated C2C12 myotubes from reaching full maturity.

Quantitative proteomics and transcriptomics addressing the estrogen receptor subtype-mediated effects in T47D breast cancer cells exposed to the phytoestrogen genistein.

The present study addresses, by transcriptomics and quantitative stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics, the estrogen receptor α (ERα) and ß (ERß)-mediated effects on gene and protein expression in T47D breast cancer cells exposed to the phytoestrogen genistein. Using the T47D human breast cancer cell line with tetracycline-dependent ERß expression (T47D-ERß), the effect of a varying intracellular ERα/ERß ratio on genistein-induced gene and protein expression was characterized. Results obtained reveal that in ERα-expressing T47D-ERß cells with inhibited ERß expression genistein induces transcriptomics and proteomics signatures pointing at rapid cell growth and migration by dynamic activation of cytoskeleton remodeling. The data reveal an interplay between integrins, focal adhesion kinase, CDC42, and actin cytoskeleton signaling cascades, occurring upon genistein treatment, in the T47D-ERß breast cancer cells with low levels of ERα and no expression of ERß. In addition, data from our study indicate that ERß-mediated gene and protein expression counteracts ERα-mediated effects because in T47D-ERß cells expressing ERß and exposed to genistein transcriptomics and proteomics signatures pointing at a clear down-regulation of cell growth and induction of cell cycle arrest and apoptosis were demonstrated. These results suggest that ERß decreases cell motility and metastatic potential as well as cell survival of the breast cancer cell line. It is concluded that the effects of genistein on proteomics and transcriptomics end points in the T47D-ERß cell model are comparable with those reported previously for estradiol with the ultimate estrogenic effect being dependent on the relative affinity for both receptors and on the receptor phenotype (ERα/ERß ratio) in the cells or tissue of interest.

Influence of cellular ERalpha/ERbeta ratio on the ERalpha-agonist induced proliferation of human T47D breast cancer cells.

Breast cancer cells show overexpression of estrogen receptor (ER) alpha relative to ERbeta compared to normal breast tissues. This observation has lead to the hypothesis that ERbeta may modulate the proliferative effect of ERalpha. This study investigated how variable cellular expression ratios of the ERalpha and ERbeta modulate the effects on cell proliferation induced by ERalpha or ERbeta agonists, respectively. Using human osteosarcoma (U2OS) ERalpha or ERbeta reporter cells, propyl-pyrazole-triol (PPT) was shown to be a selective ERalpha and diarylpropionitrile (DPN) a preferential ERbeta modulator. The effects of these selective estrogen receptor modulators (SERMs) and of the model compound E2 on the proliferation of T47D human breast cancer cells with tetracycline-dependent expression of ERbeta (T47D-ERbeta) were characterized. E2-induced cell proliferation of cells in which ERbeta expression was inhibited was similar to that of the T47D wild-type cells, whereas this E2-induced cell proliferation was no longer observed when ERbeta expression in the T47D-ERbeta cells was increased. In the T47D-ERbeta cell line, DPN also appeared to be able to suppress cell proliferation when levels of ERbeta expression were high. In the T47D-ERbeta cell line, PPT was unable to suppress cell proliferation at all ratios of ERalpha/ERbeta expression, reflecting its ability to activate only ERalpha and not ERbeta. It is concluded that effects of estrogen-like compounds on cell proliferation are dependent on the actual ERalpha/ERbeta expression levels in these cells or tissues and the potential of the estrogen agonists to activate ERalpha and/or ERbeta.