Nanog transforms NIH3T3 cells and targets cell-type restricted genes

The transcription factor Nanog is uniquely expressed in embryonic stem (ES) cells and in germ cell tumors and is important for selfrenewal. To understand the relation between this and cell transformation, we expressed Nanog in NIH3T3 cells, and these cells showed an increased growth rate and a transformed phenotype as demonstrated by foci formation and colony growth in soft agar. This suggests that Nanog possesses an oncogenic potential that may be related to the role it plays in germ cell tumors and to its function in self renewal of ES cells. We studied the transcription targets of Nanog using microarrays to identify Nanog regulated genes. The list of genes regulated by Nanog was unique for each cell type and more than 10 % of the Nanog regulated genes, including transcription factors, are primary Nanog targets since their promoters bind Nanog in ES cells. Some of these target genes can explain the transformation o

SHaploseek is a sequencing-only, high-resolution method for comprehensive preimplantation genetic testing

Abstract Recent advances in genomic technologies expand the scope and efficiency of preimplantation genetic testing (PGT). We previously developed Haploseek, a clinically-validated, variant-agnostic comprehensive PGT solution. Haploseek is based on microarray genotyping of the embryo’s parents and relatives, combined with low-pass sequencing of the embryos. Here, to increase throughput and versatility, we aimed to develop a sequencing-only implementation of Haploseek. Accordingly, we developed SHaploseek, a universal PGT method to determine genome-wide haplotypes of each embryo based on low-pass (≤ 5x) sequencing of the parents and relative(s) along with ultra-low-pass (0.2–0.4x) sequencing of the embryos. We used SHaploseek to analyze five single lymphoblast cells and 31 embryos. We validated the genome-wide haplotype predictions against either bulk DNA, Haploseek, or, at focal genomic sites, PCR-based PGT results. SHaploseek achieved > 99% concordance with bulk DNA in two families from which single cells were derived from grown-up children. In embryos from 12 PGT families, all of SHaploseek’s focal site haplotype predictions were concordant with clinical PCR-based PGT results. Genome-wide, there was > 99% median concordance between Haploseek and SHaploseek’s haplotype predictions. Concordance remained high at all assayed sequencing depths ≥ 2x, as well as with only 1ng of parental DNA input. In subtelomeric regions, significantly more haplotype predictions were high-confidence in SHaploseek compared to Haploseek. In summary, SHaploseek constitutes a single-platform, accurate, and cost-effective comprehensive PGT solution

Snord 3A: a molecular marker and modulator of prion disease progression.

Since preventive treatments for prion disease require early identification of subjects at risk, we searched for surrogate peripheral markers characterizing the asymptomatic phases of such conditions. To this effect, we subjected blood mRNA from E200K PrP CJD patients and corresponding family members to global arrays and found that the expression of Snord3A, a non-coding RNA transcript, was elevated several times in CJD patients as compared to controls, while asymptomatic carriers presented intermediate Snord3A levels. In the brains of TgMHu2ME199K mice, a mouse model mimicking for E200K CJD, Snord 3A levels were elevated in an age and disease severity dependent manner, as was the case for brains of these mice in which disease was exacerbated by copper administration. Snord3A expression was also elevated in scrapie infected mice, but not in PrP(0/0) mice, indicating that while the expression levels of this transcript may reflect diverse prion etiologies, they are not related to the loss of PrP(C)'s function. Elevation of Snord3A was consistent with the activation of ATF6, representing one of the arms of the unfolded protein response system. Indeed, SnoRNAs were associated with reduced resistance to oxidative stress, and with ER stress in general, factors playing a significant role in this and other neurodegenerative conditions. We hypothesize that in addition to its function as a disease marker, Snord3A may play an important role in the mechanism of prion disease manifestation and progression

Identification of IRF-8 and IRF-1 target genes in activated macrophages.

Interferon regulatory factor 1 (IRF-1) and IRF-8, also known as interferon consensus sequence binding protein (ICSBP), are important regulators of macrophage differentiation and function. These factors exert their activities through the formation of heterocomplexes. As such, they are coactivators of various interferon-inducible genes in macrophages. To gain better insights into the involvement of these two transcription factors in the onset of the innate immune response and to identify their regulatory network in activated macrophages, DNA microarray was employed. Changes in the expression profile were analyzed in peritoneal macrophages from wild type mice and compared to IRF-1 and IRF-8 null mice, before and following 4 h exposure to IFN-gamma and LPS. The expression pattern of 265 genes was significantly changed (up/down) in peritoneal macrophages extracted from wild type mice following treatment with IFN-gamma and LPS, while no changes in the expression levels of these genes were observed in samples of the same cell-type from both IRF-1 and IRF-8 null mice. Among these putative target genes, numerous genes are involved in macrophage activity during inflammation. The expression profile of 10 of them was further examined by quantitative RT-PCR. In addition, the promoter regions of three of the identified genes were analyzed by reporter gene assay for the ability to respond to IRF-1 and IRF-8. Together, our results suggest that both IRF-1 and IRF-8 are involved in the transcriptional regulation of these genes. We therefore suggest a broader role for IRF-1 and IRF-8 in macrophages differentiation and maturation, being important inflammatory mediators

Establishment of Homozygote Mutant Human Embryonic Stem Cells by Parthenogenesis

<div><p>We report on the derivation of a diploid 46(XX) human embryonic stem cell (HESC) line that is homozygous for the common deletion associated with Spinal muscular atrophy type 1 (SMA) from a pathenogenetic embryo. By characterizing the methylation status of three different imprinted loci (MEST, SNRPN and H19), monitoring the expression of two parentally imprinted genes (SNRPN and H19) and carrying out genome-wide SNP analysis, we provide evidence that this cell line was established from the activation of a mutant oocyte by diploidization of the entire genome. Therefore, our SMA parthenogenetic HESC (pHESC) line provides a proof-of-principle for the establishment of diseased HESC lines without the need for gene manipulation. As mutant oocytes are easily obtained and readily available during preimplantation genetic diagnosis (PGD) cycles, this approach should provide a powerful tool for disease modelling and is especially advantageous since it can be used to induce large or complex mutations in HESCs, including gross DNA alterations and chromosomal rearrangements, which are otherwise hard to achieve.</p></div

The E2F site in the common promoter region has opposite effects on <i>RAD51</i> and <i>TODRA</i>.

<p><b>A. Top:</b> Diagram of the core <i>TODRA</i> promoter region cloned into the luciferase reporter vector. <b>Bottom:</b><u>Effect of mutagenesis of the E2F binding site and E2F1 induction on the <i>TODRA</i> reporter.</u> Wild type <i>TODRA</i> luciferase (reporter) construct, or an E2F binding site mutant (E2F site mut) construct were transfected into MCF7 and U2OS cells. A <i>TODRA</i> luciferase (reporter) construct was also co-transfected with either an E2F1 expression vector or an empty vector control into serum-starved MCF7 and U2OS cells. All experiments included co-transfection with pRL-TK (to normalize for transfection efficiency). Results are depicted as the fold change in RLA compared to the WT construct transfection. Values in all experiments are means ± SE of 3–4 independent transfections performed in duplicate. ** p≤ 0.007, * p≤ 0.02, ^ p = 0.00001. <b>B. Top:</b> Diagram of the core <i>RAD51</i> promoter region cloned into the luciferase reporter vector. <b>Bottom:</b><u>Effect of mutagenesis of the E2F binding site and E2F1 induction on the <i>RAD51</i> reporter.</u> Wild type <i>RAD51</i> luciferase (reporter) construct, or an E2F binding site mutant (E2F site mut) construct were transfected into MCF7 and U2OS cells. A <i>RAD51</i> luciferase (reporter) construct was also co-transfected with either an E2F1 expression vector or an empty vector control into serum-starved MCF7 and U2OS cells. All experiments included co-transfection with pRL-TK (to normalize for transfection efficiency). Results are depicted as the fold change in RLA compared to the WT construct transfection. Values in all experiments are means ± SE of 3–4 independent transfections performed in duplicate. ** p≤ 0.007, * p≤ 0.02. <b>C. and D. Top:</b> Diagram of the <i>RAD51/TODRA</i> bidirectional promoter region cloned between the firefly and Renilla luciferase reporter genes (pBDP). <b>C.</b><u>Mutagenesis of the E2F binding site</u>. E2F site mutant (pBDP E2F site mut) or wild type bidirectional promoter constructs (pBDP) were transfected into MCF7 and U2OS cells. Results are depicted as the fold change in the mutant compared to the WT in the ratio of Firefly/Renilla luciferase activities, which represents the ratio of <i>RAD51/TODRA</i> promoter activities. Values are means ± SE of 3–6 independent transfections performed in duplicate. ** p< 0.0001. <b>D.</b><u>E2F1 overexpression</u>. pBDP activity was examined in MCF7 and U2OS cells co-transfected with the pBDP construct and either an E2F1 WT, an E2F1 trans-activation domain deletion mutant (ΔTA), or an empty expression vector. Results are depicted as the fold change between each E2F1 expression vector and the empty vector control, in the ratio of Firefly/Renilla luciferase activities, which represents the ratio of <i>RAD51/TODRA</i> promoter activities. Values are means ± SE of 3–6 independent transfections performed in duplicate. ** p< 0.0001. Additional comparisons are indicated above the bars. * p≤ 0.003.</p

<i>TODRA</i> lncRNA plays a role in a new feedback loop regulating <i>RAD51</i> expression and activity.

<p>E2F1 induction enhances <i>RAD51</i> expression (thin green arrow) while simultaneously reducing lncRNA <i>TODRA</i> expression. While E2F1 induction of <i>RAD51</i> is synergistically enhanced by TPIP (thick green arrow), E2F1 induction also reduces <i>TPIP</i> expression, possibly by affecting <i>TODRA</i> expression, as <i>TODRA</i> expression can increase <i>TPIP</i> expression. This feedback regulation of <i>RAD51</i> expression can fine-tune <i>RAD51</i> expression and HR-DSB repair. Green: Enhancement of expression/activity. Red: Suppression of expression/activity.</p

The <i>RAD51-TODRA</i> regulatory pathway in breast cancer tumors.

<p>Relationship between transcript expression levels, along the <i>RAD51-TODRA</i> regulatory pathway, in breast cancer tumors (based on data from Muggerud <i>et al</i>., 2010[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134120#pone.0134120.ref028" target="_blank">28</a>]). + positive correlation,—negative correlation. NS: not significant. All p-values are for 2-tailed analysis. Pearson correlation was used for comparison of continuous variables and Spearman correlation and t-test for non-parametric comparisons.</p><p>* Asterisks indicate gene-gene correlations that reflect perturbation of the normal pathway.</p><p>The <i>RAD51-TODRA</i> regulatory pathway in breast cancer tumors.</p

TPIP regulates <i>RAD51</i> expression and activity.

<p><b>A.</b><u>TPIP co-activates E2F1 induction of <i>RAD51</i>.</u> pRAD51-UTR with an E2F1 expression vector or an empty vector were co-transfected into serum starved MCF7 cells together with either PTEN, TPIPα, TPIPβ or an empty pEGFP-C2 based expression vector and pRL-TK (to normalize for transfection efficiency). Results are depicted as fold change in RLA compared to pRAD51-UTR alone (left bar). Values are means ± SE of 3 independent transfections performed in duplicates. ** p< 0.0001. Additional comparisons are indicated above the bars. * p = 0.002. <b>B.</b><u>E2F1 induction and endogenous <i>TPIP</i> expression</u>. Endogenous <i>TPIP</i> mRNA levels were determined using quantitative real-time RT-PCR normalized to <i>GAPDH</i>, with and without E2F1 induction in serum starved ER-E2F1 U2OS cells. E2F1 was induced by treatment with OHT for 4 hours. Values are means ± SE of 4 independent experiments. Real-time reactions were performed in duplicates. ** p< 0.00001. <b>C.</b><u>Overexpression of <i>TPIP</i> reduces HR.</u> HRind cells were transfected with an mOrange2 control vector (CV) or <i>TPIP</i> expression vector (tagged with mOrange2) and induced with Dexamethasone for 48 hours. GFP expression was measured by FACS. Results are depicted as the fold change in observed HR (as indicated by the number of GFP-positive cells among the transfected population [mOrange2 positive cells]) compared to the control vector. Values are means ± SE of 3 independent experiments performed in triplicate. ** p<0.002.</p