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

Counteracting Activities of OCT4 and KLF4 during Reprogramming to Pluripotency

Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) after overexpressing four transcription factors, of which Oct4 is essential. To elucidate the role of Oct4 during reprogramming, we investigated the immediate transcriptional response to inducible Oct4 overexpression in various somatic murine cell types using microarray analysis. By downregulating somatic-specific genes, Oct4 induction influenced each transcriptional program in a unique manner. A significant upregulation of pluripotent markers could not be detected. Therefore, OCT4 facilitates reprogramming by interfering with the somatic transcriptional network rather than by directly initiating a pluripotent gene-expression program. Finally, Oct4 overexpression upregulated the gene Mgarp in all the analyzed cell types. Strikingly, Mgarp expression decreases during the first steps of reprogramming due to a KLF4-dependent inhibition. At later stages, OCT4 counteracts the repressive activity of KLF4, thereby enhancing Mgarp expression. We show that this temporal expression pattern is crucial for the efficient generation of iPSCs

Nanog induces hyperplasia without initiating tumors

Though expression of the homeobox transcription factor Nanog is generally restricted to pluripotent cells and early germ cells, many contradictory reports about Nanog's involvement in tumorigenesis exist. To address this, a modified Tet-On system was utilized to generate Nanog-inducible mice. Following prolonged Nanog expression, phenotypic alterations were found to be restricted to the intestinal tract, leaving other major organs unaffected. Intestinal and colonic epithelium hyperplasia was observed—intestinal villi had doubled in length and hyperplastic epithelium outgrowths were seen after 7 days. Increased proliferation of crypt cells and downregulation of the tumor suppressors Cdx2 and Klf4 was detected. ChIP analysis showed physical interaction of Nanog with the Cdx2 and Klf4 promoters, indicating a regulatory conservation from embryonic development. Despite downregulation of tumor suppressors and increased proliferation, ectopic Nanog expression did not lead to tumor formation. We conclude that unlike other pluripotency-related transcription factors, Nanog cannot be considered an oncogene

<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

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

<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

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

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