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

Whole genome view of Cytoscan SNP Array data.

<p>Genome wide SNP array results obtained from (A) reference DNA from a male with a normal karyotype (46; XY); and (B) SZ-SMA5 HESC line DNA. The X axis represents chromosomes 1–22, X and Y. (I) The Y axis represents the copy number, determined by the log2 ratio (grey dots) on the left side of the graph, and it's smoothed ratio (red line) on the right. The expected copy number is 2 for autosomal chromosomes (log2 of 0 and smooth signal of 2). The log2 ratio and the smooth signal are determined from both the nonpolymorphic copy number probes and the polymorphic SNP probes. (II) The Y-axis corresponds to homozygote calls (AA or BB) and heterozygote calls (AB). Allele peaks of 1, 0, and -1 indicate a copy number of two, while allele peaks of 0.5 and -0.5 indicate a copy number of one. Allele peaks are calculated from SNP probes. The distinction between XY (reference DNA) and XX (SZ-SMA5) cells is clearly illustrated by the difference in X chromosome copy number. In addition, the overall 0.49% inherent heterozygote call error rate in SZ-SMA5 is below even the expected array genotyping error of ~1% (as determined by dividing the number of heterozygous calls by the total number of SNP probes on the array). Therefore, these data indicate that SZ-SMA5 features a completely homozygous diploid genome. (C) Fraction of SNP heterozygote calls in WT male reference and SZ-SMA5 DNA. Chromosomes are indicated in the X axis and the Y axis indicates the fraction of heterozygous SNP calls per total SNP calls on each chromosome.</p

Characterization of SZ-SMA5.

<p>(A) Expression of undifferentiated cell specific markers by immunostaining for <i>OCT4</i> (red nuclear staining, merged onto Hoechst (blue)), for the cell surface marker <i>Tra 1–60</i> (red, merged onto Hoechst (blue)); and (B) for alkaline phosphatase activity (AP). (C) Expression of undifferentiated cell specific markers: <i>OCT4</i>, <i>REX1</i>, <i>NANOG</i> and <i>SOX2</i> in SZ-SMA5 and a wild-type (WT) HESC control by RT-PCR. <i>GAPDH</i> expression served as a loading control. (D) Teratoma sections stained by H&E from SZ-SMA5 demonstrating multi-cellular structures derived from the three different embryonic germ layers. (E) A representative normal 46(XX) karyotype in SZ-SMA5 as identified by Giemsa staining of 20 different spreads of metaphase chromosomes.</p

<i>TODRA</i>, a lncRNA at the <i>RAD51</i> Locus, Is Oppositely Regulated to <i>RAD51</i>, and Enhances RAD51-Dependent DSB (Double Strand Break) Repair

<div><p>Expression of <i>RAD51</i>, a crucial player in homologous recombination (HR) and DNA double-strand break (DSB) repair, is dysregulated in human tumors, and can contribute to genomic instability and tumor progression. To further understand <i>RAD51</i> regulation we functionally characterized a long non-coding (lnc) RNA, dubbed <i>TODRA</i> (<u>T</u>ranscribed in the <u>O</u>pposite <u>D</u>irection of <u><i>RA</i></u><i>D51</i>), transcribed 69bp upstream to <i>RAD51</i>, in the opposite direction. We demonstrate that <i>TODRA</i> is an expressed transcript and that the <i>RAD51</i> promoter region is bidirectional, supporting <i>TODRA</i> expression (7-fold higher than RAD51 in this assay, p = 0.003). <i>TODRA</i> overexpression in HeLa cells induced expression of <i>TPIP</i>, a member of the TPTE family which includes PTEN. Similar to PTEN, we found that TPIP co-activates E2F1 induction of <i>RAD51</i>. Analysis of E2F1's effect on the bidirectional promoter showed that E2F1 binding to the same site that promotes <i>RAD51</i> expression, results in downregulation of <i>TODRA</i>. Moreover, <i>TODRA</i> overexpression induces HR in a RAD51-dependent DSB repair assay, and increases formation of DNA damage-induced RAD51-positive foci. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates <i>RAD51</i> and <i>TODRA</i>: increased <i>RAD51</i> expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and <i>HER2</i> amplification (r = 0.41, p = 0.001)), correlates as expected with lower <i>TODRA</i> and higher <i>E2F1</i> expression. However, although E2F1 induction resulted in <i>TPIP</i> downregulation in cell lines, we find that <i>TPIP</i> expression in tumors is not reduced despite higher <i>E2F1</i> expression, perhaps contributing to increased <i>RAD51</i> expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the <i>TODRA</i> lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy. Importantly, gene expression in breast tumors supports our finding that E2F1 oppositely regulates RAD51 and TODRA: increased RAD51 expression, which is associated with an aggressive tumor phenotype (e.g. negative correlation with positive ER (r = -0.22, p = 0.02) and positive PR status (r = -0.27, p<0.001); positive correlation with ki67 status (r = 0.36, p = 0.005) and HER2 amplification (r = 0.41, p = 0.001)), correlates as expected with lower TODRA and higher E2F1 expression. However, although E2F1 induction resulted in TPIP downregulation in cell lines, we find that TPIP expression in tumors is not reduced despite higher E2F1 expression, perhaps contributing to increased RAD51 expression. Our results identify TPIP as a novel E2F1 co-activator, suggest a similar role for other TPTEs, and indicate that the TODRA lncRNA affects RAD51 dysregulation and RAD51-dependent DSB repair in malignancy.</p></div

Methylation levels at <i>H19</i>, <i>SNRPN</i> and <i>MEST</i> imprinted loci.

<p>Bisulfite single colony sequencing was performed on 3 different imprinted regions (<i>H19</i>, SNRPN and <i>MEST</i>) to determine methylation levels in normal (WT) and SZ-SMA5 HESC lines. Each line represents a single DNA molecule. Full circles correspond to methylated CpGs while empty circles represent unmethylated CpGs.</p

<i>TODRA</i> promotes homologous recombination repair of DSBs.

<p><b>A.</b><u>Schematic representation of the HRind cell system</u><b>.</b> The mCherry-<i>ISce</i>I-GR (Glucoroticoid Receptor) endonuclease is cytoplasmic. Upon addition of Dexamethasone, it rapidly translocates into the nucleus generating a DSB at the <i>ISce</i>I site in the DR-GFP cassette. The DSB can be repaired either by NHEJ (non-homologous end-joining) or HR, but only HR repair reconstitutes functional GFP (green nucleus) from DR-GFP. <b>B.</b><u>Overexpression of <i>TODRA</i> induces RAD51-dependent HR.</u> HRind cells were transfected with an empty vector (EV) or <i>TODRA</i> minigene 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) compared to the empty vector. Values are means ± SE of 3 independent experiments performed in triplicate. * p< 0.04. <b>C.</b><u>Overepxression of <i>TODRA</i> elevates DNA damage-induced RAD51 foci formation.</u> U2OS cells were transfected with an empty vector (EV) or the <i>TODRA</i> minigene. 48 hrs. post transfection half of each culture was treated for 1 hr. with the DNA damaging agent phleomycin (10μg/ml). Medium was then replaced in all cultures, releasing treated cells from phleomycin exposure. γH2AX and RAD51 foci were imaged either immediately (0 hours) or 6 hours after removal of phleomycin and medium exchange. <b>Top:</b> A representative image of γH2AX (green) and RAD51 (red) foci in empty vector (EV) and <i>TODRA</i> transfected cells 6 hours after removal of phleomycin. DAPI (blue signal in merged images) was used for counterstaining. Scale bars = 10 μm. <b>Bottom:</b> The number of RAD51-positive foci was normalized as the fraction of γH2AX-positive foci per cell and averaged across all samples in each condition. Cells were treated with phleomycin, as indicated, and fixed at the indicated time points post-treatment. Results are depicted as the fold change in the fraction of RAD51 foci in cells overexpressing <i>TODRA</i> compared to the empty vector. Values are means ± SE of 3 independent experiments. * p≤ 0.03.</p

E2F1 induction oppositely affects endogenous <i>RAD51</i> and <i>TODRA</i> expression.

<p><b>A.</b><u>E2F1 induction results in E2F1 binding of the <i>RAD51/TODRA</i> promoter</u>. E2F1 expression was induced in serum starved ER-E2F1 U2OS cells (stably transfected with a constitutively expressed ER-E2F1 fusion protein which upon ligand-dependent activation translocates from the cytoplasm to the nucleus) by treatment with OHT for 8 hours. <i>RAD51/TODRA</i> promoter occupancy was measured with a ChIP assay using E2F1 antibodies (Ab) in lysates of either OHT treated or untreated cells. Real-time PCR was performed to quantitate the <i>RAD51/TODRA</i> template captured by the E2F1 Ab. Promoter occupancy is expressed as fold change relative to that observed in untreated cells. Values are means ± SE of 3 ChIP independent experiments. Real-time reactions were performed in triplicates. ^ p = 0.01. A representative gel of the promoter region PCR amplification products is shown on the left (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134120#pone.0134120.s002" target="_blank">S2 Fig</a>). <b>B.</b><u>E2F1 induction and endogenous <i>RAD51</i> and <i>TODRA</i> transcription</u>. <i>RAD51</i> and <i>TODRA</i> transcript levels were determined after E2F1 induction (see above), using quantitative real-time RT-PCR normalized to <i>GAPDH</i>. Results are depicted as the fold change in either <i>RAD51</i> or <i>TODRA</i> transcript levels compared to non-treated cells (time 0). Values are means ± SE of 3–4 independent experiments. Real-time reactions were performed in duplicates. + p≤ 0.04, * p≤ 0.004.</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