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

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

Transcriptional analysis of the <i>RAD51/TODRA</i> region.

<p><b>A.</b><u><i>TODRA</i> transcript:</u><b>Top:</b><u>Schematic representation of the predicted <i>TODRA</i> (<i>AK125393</i>) gene</u>, as described in the UCSC genome browser. Light grey shaded rectangles depict <i>TODRA</i> exons, the dark grey rectangle depicts <i>RAD51</i> exon 1, transcribed in the opposite direction. <b>Bottom:</b><u>Results of <i>TODRA</i> transcript analysis.</u> 5’RACE using capped HeLa mRNA, identified one transcription start site (full arrowhead, +1 corresponds to chr. 15: 40987374, hg19), and 3’RACE identified several possible transcription termini (arrows). The most 5’ end of <i>RAD51</i> identified using 5’RACE is also shown (empty arrowhead, +1 corresponds to chr. 15: 40987303, hg19). Black bars beneath the diagram indicate confirmed regions of unidirectional transcription determined using strand specific primers for reverse transcription from both HeLa and MCF7 cells. <b>B.</b><u>Splicing of <i>TODRA</i> exons 1 and 2</u> is demonstrated in the representative gel. Lane M: pUC Mix Marker, (Fermentas), Lanes 1&2: <i>TODRA</i> strand specific RT-PCR products (F primer located in exon 1, R primer in exon 2). Expected size of product in genomic DNA: 696bp, Expected size of spliced transcript: 480bp, as observed in lanes 1 (cDNA prepared from HeLa cells) and 2 (cDNA prepared from MCF7 cells). <b>C.</b><u>The <i>RAD51/TODRA</i> region supports transcription in both directions.</u><b>Top:</b><u>Schematic representation of the <i>RAD51</i> and <i>TODRA</i> promoter regions</u> and the fragments cloned into luciferase promoter constructs. <b>Bottom:</b><u><i>TODRA</i> putative promoter activity</u>. MCF7 cells were co-transfected with the promoter-less pGL3-basic, pRAD51-UTR or pTODRA and pRL-TK (to normalize for transfection efficiency). Results are shown as fold increase in RLA (relative luciferase activity), compared to pGL3-basic. Values are means ± SE of 4–5 independent transfections performed in duplicates. * p< 0.002, ** p< 0.0001.</p

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

Identification of Snord3A and Aldh1A1 as disease specific markers.

<p>a: Gene expression heat map representing the levels of transcripts expressed in blood from E200KCJD patients, carriers and non-carriers control. b: Snord3A expression level in microarrays of patients and healthy mutation carriers as compared to non-carrier controls. C: Aldh1A1 expression level in patients and healthy mutation carriers as compared to non-carrier controls (P value <0.005).</p