STELLA Facilitates Differentiation of Germ Cell and Endodermal Lineages of Human Embryonic Stem Cells

Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES) cells and in primordial germ cells (PGCs). In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA)-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells

Molecular mechanisms of pluripotency and reprogramming

Pluripotent stem cells are able to form any terminally differentiated cell. They have opened new doors for experimental and therapeutic studies to understand early development and to cure degenerative diseases in a way not previously possible. Nevertheless, it remains important to resolve and define the mechanisms underlying pluripotent stem cells, as that understanding will impact strongly on future medical applications. The capture of pluripotent stem cells in a dish is bound to several landmark discoveries, from the initial culture and phenotyping of pluripotent embryonal carcinoma cells to the recent induction of pluripotency in somatic cells. On this developmental time line, key transcription factors, such as Oct4, Sox2 or Nanog, have been revealed not only to regulate but also to functionally induce pluripotency. These early master regulators of development control developmental signalling pathways that affect the cell cycle, regulate gene expression, modulate the epigenetic state and repair DNA damage. Besides transcription factors, microRNAs have recently been shown to play important roles in gene expression and are embedded into the regulatory network orchestrating cellular development. However, there are species-specific differences in pluripotent cells, such as surface marker expression and growth factor requirements. Such differences and their underlying developmental pathways require clear definition and have major impacts on the preclinical test bed of pluripotent cells

Aza-deoxycytidine induces apoptosis or differentiation via DNMT3B and targets embryonal carcinoma cells but not their differentiated derivatives

Background: Teratocarcinoma is a malignant male germ cell tumour, which contains stem cells and differentiated cancer tissues. DNMT3B has been shown to be highly expressed in human teratocarcinoma stem cells, and to mediate cytotoxicity of Aza-deoxycytidine (Aza-dC) in a pluripotent stem cell line NTERA2. Methods: We have established DNMT3B or POU5F1 (hereafter referred to as OCT4) knockdown in teratocarcinoma stem cells N2102Ep and TERA1 and in the pluripotent NTERA2 by a doxycycline-inducible system, and tested the cytotoxicity induced by Aza-dC. Results: Silencing of DNMT3B led to apoptosis of human teratocarcinoma stem cells N2102Ep and TERA1. Further, we found that induction of apoptosis or differentiation in NTERA2 and human embryonic stem cells by Aza-dC requires DNMT3B. To test whether Aza-dC inhibits proliferation of differentiated teratocarcinoma cells, we depleted OCT4 expression in N2102Ep and TERA1 cells treated with Aza-dC. Treatment with Aza-dC reduced cell number of differentiated cells to a lesser extent than their undifferentiated parental stem cells. Moreover, in contrast to the stem cells, Aza-dC failed to induce apoptosis of differentiated cells. Conclusions: Our finding suggests that DNMT3B acts as an antiapoptotic gene in teratocarcinoma stem cells, and mediates apoptosis and differentiation of human pluripotent stem cells induced by Aza-dC, and that Aza-dC specifically induces apoptosis of teratocarcinoma stem cells

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.Oncogene advance online publication, 25 July 2016; doi:10.1038/onc.2016.260

RNA Binding to CBP Stimulates Histone Acetylation and Transcription

CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP—a regulatory motif unique to CBP/p300—allows RNA to stimulate CBP’s HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes

Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.This work was supported by FEDER and FCT – Fundação para a Ciência e a Tecnologia (contract PEst-OE/EQB/LA0023/2011_ research line: Systems and Synthetic Biology; PhD grant to A.M. – SFRH/BD/37012/2007, and PD grants to S.S. – SFRH/BPD/75483/2010 and C.C. – SFRH/BPD/ 81220/2011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.info:eu-repo/semantics/publishedVersio

New Insights Into the Long Non-coding RNA SRA:Physiological Functions and Mechanisms of Action

Long non-coding RNAs (lncRNA) are emerging as new genetic/epigenetic regulators that can impact almost all physiological functions. Here, we focus on the long non-coding steroid receptor RNA activator (SRA), including new insights into its effects on gene expression, the cell cycle, and differentiation; how these relate to physiology and disease; and the mechanisms underlying these effects. We discuss how SRA acts as an RNA coactivator in nuclear receptor signaling; its effects on steroidogenesis, adipogenesis, and myocyte differentiation; the impact on breast and prostate cancer tumorigenesis; and, finally, its ability to modulate hepatic steatosis through several signaling pathways. Genome-wide analysis reveals that SRA regulates hundreds of target genes in adipocytes and breast cancer cells and binds to thousands of genomic sites in human pluripotent stem cells. Recent studies indicate that SRA acts as a molecular scaffold and forms networks with numerous coregulators and chromatin-modifying regulators in both activating and repressive complexes. We discuss how modifications to SRA's unique stem-loop secondary structure are important for SRA function, and highlight the various SRA isoforms and mutations that have clinical implications. Finally, we discuss the future directions for better understanding the molecular mechanisms of SRA action and how this might lead to new diagnostic and therapeutic approaches

Functional study of DNMT3B in human pluripotent and nullipotent stem cells

Human embryonic stem (ES) cells and embryonal carcinoma (EC) cells are the stem cells derived from human blastocysts and teratocarcinoma germ cell tumors, respectively. The majority of human EC cell lines have been shown to be nullipotent stem cells, which are not competent to be induced to differentiation both in vitro and in vivo compared with their pluripotent counterparts. However, by knocking down OCT4 it has been shown to induce differentiation of nullipotent EC cells. In this thesis, gene expression analysis has shown that these two stem cell types are different in term of inner cell mass- and early germline-specific gene expression profiles. STELLA was found to be up-regulated in EC compared with ES cells. However, an ectopic over-expression of STELLA in ES cells did not promote self-renewal. Rather, it facilitated germline and endodermal differentiation of the stem cells. DNMT3B was observed to be highly expressed in human nullipotent EC cells, and to be down-regulated during differentiation of ES cells. Silencing of DNMT3B by shRNAs did not alter the stem cell state of either ES or EC cells, but led to a reduction of differentiation of ES cells. In addition, reprogramming of differentiated cells in which DNMT3B had been knocked down, to produce induced pluripotent stem cells, was more efficient than reprogramming differentiated cells expressing DNMT3B. These results suggest that DNMT3B promote the differentiation capacity of pluripotent stem cells. Inhibition of DNA methylation by Aza-deoxycytidine (Aza-dC) resulted in differentiation and apoptosis of pluripotent ES and EC cells. However, knockdown of DNMT3B antagonized differentiation and apoptosis induced by Aza-dC, suggesting that the ability of the stem cells to undergo these two fates is DNMT3B-dependent. Moreover, the differentiated EC cells, induced to differentiate by OCT4-knockdown, had a reduced apoptotic response to Aza-dC, implying a stem cell-specific induction of apoptosis by the inhibition of DNA methylation. Since the inhibition of DNA methylation does not lead to differentiation of nullipotent EC cells, DNA methylation might not be important for maintenance of the nullipotent state. To test whether other epigenetic mechanisms are nevertheless involved in the maintenance of nullipotency, N21 02Ep differentiated nUllipotent EC cells generated by OCT4 silencing were reprogrammed to achieve a pluripotent state by transfection with a vector encoding the Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC. Stem cells derived from this reprogramming show a potential to down-regulate stem cell markers such as SSEA-3, and up-regulate differentiation markers for example A2B5, PAX6 and TUJ 1 during retinoic acid-induced differentiation, whereas their nullipotent parental cells did not. These results suggest that epigenetic reprogramming leads to an acquisition of a more relaxed stem cell state of the reprogrammed N2102Ep cells, and that epigenetics is a factor implicated in the maintenance of nUllipotency of human EC cells. Altogether, the finding in this thesis implicates epigenetic factors, in particular DNMT3B, in regulation of stem cell fates and states of human ES and EC cells.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

CTCF Recruits Centromeric Protein CENP-E to the Pericentromeric/Centromeric Regions of Chromosomes through Unusual CTCF-Binding Sites

The role of CTCF in stabilizing long-range interactions between chromatin sites essential for maintaining nuclear architecture is well established. Most of these interactions involve recruitment of the cohesin complex to chromatin via CTCF. We find that CTCF also interacts with the centromeric protein CENP-E both in vitro and in vivo. We identified CTCF sites in pericentric/centromeric DNA and found that, early in mitosis, CTCF binds and recruits CENP-E to these sites. Unlike most known CTCF genomic sites, the CTCF-binding sites in the pericentric/centromeric regions interact strongly with the C-terminal fingers of CTCF. Overexpression of a small CENP-E fragment, targeted to these CTCF sites, results in a delay in alignment of some chromosomes during mitosis, suggesting that the recruitment of CENP-E by CTCF is physiologically important. We conclude that CTCF helps recruit CENP-E to the centromere during mitosis and that it may do so through a structure stabilized by the CTCF/CENP-E complex

The lncRNA SRA directly interacts with TrxG and PRC2 complexes.

<p>(A) RNA pull down experiment using nuclear extract of human pluripotent stem cells NTERA2 followed by western blotting of indicated proteins. (B) Purified recombinant TrxG and PRC2 complexes were used for <i>in vitro</i> RNA pull down experiment and western blotting. Below: RT-PCR of antisense or sense SRA purified from <i>in vitro</i> pull down reactions. (C) The TrxG core component WDR5 and the PRC2 component EED and SUZ12 directly associate with SRA. Purified recombinant proteins were used for RNA pull down experiment and western blotting. (D) 5’ and 3’ domains of SRA are preferential binding regions for TrxG and PRC2 complexes, respectively. Right: RT-PCR of full length, 5’ and 3’ domains of SRA from pull down reactions; these three panels represent independent experiments and should not be compared. (E) SRA can tether TrxG and PRC2 complexes. Co-immunoprecipitation (Co-IP) was performed using purified TrxG and PRC2 complexes in the presence of antisense or sense SRA. Left: Co-IP using RBBP5 antibody. Right: Co-IP using EZH2 antibody. (F) SRA mediates interaction between TrxG and PRC2 <i>in vivo</i>. Co-IP was performed by using nuclear extract of scrambled RNA control and <i>SRA</i> knockdown NTERA2 cells. All experiments were performed as at least two independent replicates. The inputs were used at 10% of the samples.</p