Fbx15 Is a Novel Target of Oct3/4 but Is Dispensable for Embryonic Stem Cell Self-Renewal and Mouse Development

Abstract

Embryonic stem (ES) cells are derived from mammalian blastocysts and maintain pluripotency, an ability to differentiate into all types of somatic and germ cells (32). Another important property of ES cells is their robust and infinite growth equivalent to tumor cells despite their normal karyotype. ES cells were developed from mouse blastocysts in 1981 (8, 15) and have been extensively used to generate knockout mice. Human ES cells were established in 1998 (33) and are considered promising sources for cell transplantation therapy.POU transcription factor Oct3/4 is expressed specifically in pluripotent cells, including ES cells, early embryos, and germ cells (27,31). Targeted disruption of the Oct3/4 gene in the mouse results in early embryonic lethality (21). The inner cellular mass of Oct3/4-null blastocysts differentiates exclusively into trophoblasts. Furthermore, conditional deletion of Oct3/4 in ES cells leads to spontaneous differentiation into trophectoderm (25), demonstrating that Oct3/4 is essential for selfrenewal of ES cells and mouse early development.Only a few Oct3/4 target genes have been identified. These include FGF-4 (4) and Rex-1 (2), in which Oct3/4 binds to an octamer motif, ATT(T/A)GCAT, located in regulatory elements. In FGF-4, SRY-related transcription factor Sox2 binds to a motif adjacent to the octamer sequence and synergistically activates transcription (5). In Rex-1, hypothetical factor ROX1 functions in a similar manner (2). It is not clear whether synergetic interaction with other transcription factors is common among target genes. Even consensus nucleotide sequences of Oct3/4-binding sites have not been fully determined. For example, the Oct3/4-binding site in UTF1 is one nucleotide different from the octamer sequence (22). Furthermore, it remains largely unknown how Oct3/4 maintains self-renewal of ES cells. Identification of novel Oct3/4 target genes is crucial to answering these questions.In this study, we utilized expression analyses, reporter gene analyses, and a gel mobility shift assay to demonstrate that Fbx15, which encodes an F-box-containing protein (35), is a novel target of Oct3/4. We also performed gene-targeting experiments to study physiological functions of Fbx15 in selfrenewal of ES cells, mouse development, and fertility. 467, 318, 198, 408, 239, 400, 453, 109,16, 523, 161, 483, 258, 264, 419, 529, 327, 411, 417, 418, 399, 196, 271, 255, 495, 101, 98, 351, 416, 321, 251, 412, 379, 549, 329, 265, 449, 328, 516, 320, 436, 427, 297, 366, 390, 315, 228, 277, 292, 284, 285, and 30 (a total of 1,328,835 entries). MATERIALS AND METHODS DigitalCell culture. The RF8 (16), JI (13), CGR8 (20), and MG1.19 (9) ES cell lines were cultured as previously described. Differentiation of ES cells was induced with retinoic acid as previously described (36). NIH 3T3 cells were cultured with Dulbecco's modified Eagle medium (Sigma) containing 10% fetal bovine serum (Sanko Junyaku, Tokyo, Japan) and maintained at 37°C with 5% CO 2 .

Induction of pluripotency by defined factors

“…Since Fbx15 is encoded by ECAT3 and is only expressed in ES cells, but not in somatic cells like MEFs, the Fbx15 neo/neo MEFs are sensitive to G418, whereas Fbx15 neo/neo ES cells were resistant to the antibotics. 35) When each of the 24 candidates was introduced into Fbx15 neo/neo MEFs by means of retroviruses, no G418-resistant colonies emerged. However, when all 24 retroviruses were transduced at once, we obtained G418-resistant colonies, which were morphologically similar to mouse ES cells (Fig.…”

“…Even in the absence of fbx15 expression, ES cells can be established and remain pluripotent and self-renew. 35) On the other hand, Nanog and Oct3/4 are essential for ES cell fate. If the expression of Oct3/4 is suppressed by only half, the ES cells differentiate into trophectoderm.…”

A challenge for regenerative medicine: Proper genetic programming, not cellular mimicry

“…In the initial study, reactivation of the endogenous Fbx15 gene was used to identify and select iPS cells (Takahashi and Yamanaka, 2006). The Fbx15 gene is a nonessential gene, which is expressed by ES cells, but repressed after ES cells undergo differentiation (Tokuzawa et al, 2003). Despite the similarities between ES cells and Fbx15 reactivated iPS cells (Fbx15-iPS), they were far from identical.…”

Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells

“…Second, well-known target genes of the Yamanaka factors were found in our binding lists; the different probe hits of each Yamanaka factor target gene and their relative position to the transcription start-site of the corresponding gene were shown in Supplementary information, Table S1. For example, the earlier-identified Oct4 targets, including POU5F1, SOX2, FBXO15, ZIC3, GJA1, SALL4, CDX2, GDF3, DPPA3, UTF1, and FZD5 [17][18][19][20][21][22][23][24][25][26][27][28], were all included in our list of genes occupied by Oct4 (Supplementary information, Table S1). Third, we have also validated several of the predicted target loci of the Yamanaka factors by ChIP-PCR assays using primer sets containing or adjacent to the positive probes, and all tested target loci were significantly enriched over the negative controls (Supplementary information, Figure S2).…”