Zfp296 Is a Novel, Pluripotent-Specific Reprogramming Factor

Expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is sufficient to reprogram somatic cells into induced pluripotent stem (iPSCs). However, this process is slow and inefficient compared with the fusion of somatic cells with embryonic stem cells (ESCs), indicating that ESCs express additional factors that can enhance the efficiency of reprogramming. We had previously developed a method to detect and isolate early neural induction intermediates during the differentiation of mouse ESCs. Using the gene expression profiles of these intermediates, we identified 23 ESC-specific transcripts and tested each for the ability to enhance iPSC formation. Of the tested factors, zinc finger protein 296 (Zfp296) led to the largest increase in mouse iPSC formation. We confirmed that Zfp296 was specifically expressed in pluripotent stem cells and germ cells. Zfp296 in combination with OSKM induced iPSC formation earlier and more efficiently than OSKM alone. Through mouse chimera and teratoma formation, we demonstrated that the resultant iPSCs were pluripotent. We showed that Zfp296 activates transcription of the Oct4 gene via the germ cell–specific conserved region 4 (CR4), and when overexpressed in mouse ESCs leads to upregulation of Nanog expression and downregulation of the expression of differentiation markers, including Sox17, Eomes, and T, which is consistent with the observation that Zfp296 enhances the efficiency of reprogramming. In contrast, knockdown of Zfp296 in ESCs leads to the expression of differentiation markers. Finally, we demonstrated that expression of Zfp296 in ESCs inhibits, but does not block, differentiation into neural cells

Timing and efficiency of iPSC colony formation.

<p>NSCs were infected with either OSKM+<i>Zfp296</i> or with OSKM+empty vector. GFP-positive colonies from 30 fields from an automated microscope were counted in three independent replicates for each of the days shown (A). SSEA1-positive colonies from 60 fields from an automated microscope were counted in three independent replicates for each of the days shown (B). Mean and standard deviations are shown, and <i>P</i> values were calculated with t-tests.</p

Oct4 promoter methylation.

<p>Bisulfite sequencing results assessing the DNA methylation status of the <i>Oct4</i> promoter is shown for the indicated cell types. OSKM = <i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, and <i>c-Myc</i>; ESCs = embryonic stem cells; NSCs = neural stem cells. Open and filled circles represent unmethylated and methylated CpGs, respectively.</p

Pluripotency of iPSCs induced with OSKM+<i>Zfp296</i>.

<p>An iPSC line induced with OSKM+<i>Zfp296</i> is shown in phase (A), DAPI (B), Oct4 immunostained (C), or Oct4-GFP (D). Teratomas formed with this iPSC line were composed of cartilage (mesoderm; E), neural rosettes (ectoderm; F), and epithelia (endoderm; G). A chimera formed with this iPSC line is shown (H), and offspring from this chimera carried the GFP transgene, which originated from the iPSCs (I).</p

<i>Zfp296</i> knockdown in ESCs.

<p>Lentiviral shRNA constructs were packaged and used to infect ESCs. Puromycin was added to select for infected cells beginning on day 4. Efficiency of <i>Zfp296</i> knockdown is shown on the indicated days after infection (A). Quantitative RT-PCR results are shown for the indicated genes on day 7 after infection (B).</p

Candidate reprogramming factor testing results.

<p>Number of <i>Oct4-GFP</i>–positive iPSC colonies formed by NSCs infected with OSKM plus the designated factor relative to OSKM plus an empty retroviral vector. Mean and standard deviations are shown, and <i>p</i> values were calculated with t-tests.</p

<i>Zfp296</i> activates transcription from the <i>Oct4</i> promoter.

<p><i>Zfp296</i> and <i>Nanog</i> were tested by luciferase assays for transactivation of <i>Oct4</i> promoter fragments (A). The <i>P</i> value for <i>Zfp296</i>-induced transcription from CR4 compared with control was less than 0.1, as shown. CR1, CR2, CR3, and CR4 = evolutionary conserved regions 1, 2, 3, and 4, respectively, as defined by Nordhoff <i>et alia </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034645#pone.0034645-Nordhoff1" target="_blank">[20]</a>. ESCs were induced to express <i>Zfp296</i> for 18 hours followed by quantitative RT-PCR to assess the expression of transgenic <i>Zfp296</i> (B) and indicated marker genes (C) compared with uninduced cells (B). Expression of indicated marker genes on day 7 of neural differentiation in the presence of induced <i>Zfp296</i> expression relative to uninduced cells (D).</p

Candidate reprogramming factors.

<p>The 23 candidate reprogramming factors are listed, along with their expression, in differentiated ESCs according to the original array data. Also listed is the cDNA clone used for subcloning into the pMX retroviral expression vectors and the average number of <i>Oct4-GFP</i>–positive iPSC colonies obtained after infection of NSCs with the expression vector in combination with OSKM.</p>1<p>First initial signifies either IMAGE (I) or Riken (R). Indicated genes were isolated by RT-PCR on ESC RNA.</p>2<p>Tested on NSCs in combination with Oct4, Sox2, Klf4 and c-Myc.</p>3<p>Qkf cDNA clone was a generous gift from Dr. Tim Thomas.</p>4<p>PRDM14 was combined from two Riken clones: 6030400A03 and C330011M19 using an internal EcoRI site.</p