Oct25 represses transcription of nodal/activin target genes by interaction with signal transducers during Xenopus gastrulation

Abstract

The balance between differentiation signals and signals maintaining the undifferentiated state of embryonic cells ensures proper formation of germ layers. The nodal/activin pathway represents one of the major signaling chains responsible for the differentiation of embryonic cells into mesodermal and endodermal germ layers, while Oct4 is one of the major players in the maintenance of an undifferentiated state. Here we show that Oct25, an Oct4 homologue in Xenopus, antagonizes the activity of nodal/activin signaling by inhibiting the transcription of its target genes, Gsc and Mix2. The inhibitory effect is achieved by forming repression complexes on the promoters of Gsc and Mix2 between Oct25 and the signal transducers of the nodal/activin pathway, WBSCR11, FAST1, and Smad2. We have analyzed the significance of the Oct binding site for its inhibitory effect within the Gsc promoter. Albeit VP16-Oct25 fusion protein demonstrated a stimulating effect and EVE-Oct25 revealed a repression effect on an artificial reporter that is composed of eight repeats of Oct binding motifs, both fusions, like wild-type Oct25, inhibited mesendoderm formation and the activity of Gsc and Mix2 promoters. These results suggest that the regulatory effect of Oct25 on the expression of Gsc and Mix2 is mediated by specific protein/protein interactions. Furthermore, we demonstrate that histone deacetylase activities are not required for the inhibitory effect of Oct25. Our results provide a novel view in that Oct25 controls the nodal/activin pathway and thus maintains the undifferentiated state of embryonic cells in preventing them from premature differentiation.

FIGURE 1.

Analysis of Gsc and Mix2 transcription in response to Oct25 overexpression or knockdown. A–C, whole mount in situ hybridization (A) or real-time RT-PCR in whole embryos (B and C) reveal an inhibition of Gsc and Mix2 by Oct25 gain of function and a dose-dependent increase in transcription of both genes by Oct25 loss of function. D, real-time RT-PCR shows that transcription of mesodermal and endodermal markers Xbra, Gsc, Xsox17a, and Mix2 is strongly enhanced in animal caps from embryos injected with Xnr1 mRNA, but is severely inhibited by co-injection with Oct25 mRNA.

FIGURE 2.

Physical interactions between Oct25 and FAST1, Smad2, or XWBSCR11. A, GST pull-down assays reveal that Oct25 interacts with FAST1 (and vice versa), XWBSCR11, and Smad2. B, in transfected cells, Flag-tagged Oct25 formed complexes with HA-tagged Smad2, XWBSCR11, or FAST1 as displayed by precipitation with an anti-HA antibody.

FIGURE 3.

In vitro binding of Oct25 to Gsc and Mix2 promoters. A, increasing amounts of a GST-tagged XWBSCR11 fusion protein bind to an oligonucleotide containing the DE of the Gsc promoter (position -239 to -198), while GST itself does not show any binding. B, increasing amounts of Oct25 and GST-XWBSCR11 form a complex on the DE as displayed by a supershift. C, FAST1 specifically binds to an oligonucleotide containing the PE of the Gsc promoter (position -145 to -98) with a FAST1 binding motif. D, Oct25 forms a complex with FAST1 on the PE as revealed by supershifts. E, Oct25 binds to an oligonucleotide (position -1034 to -989) of the Gsc promoter containing an octamer-like motif. F, incubation of this region of the Gsc promoter with Oct25 and increasing amounts of GST-FAST1 fusion protein leads to a supershift. G, FAST1 interacts with the ARE of the Mix2 promoter (position -215 to -166) that contains a FAST1 binding motif. H, supershift generated with GST-FAST1 and increasing amounts of Oct25. The sequences corresponding to the regions of Gsc and Mix2 promoters used for EMSA and supershift assays are listed below each panel and protein binding motifs for FAST1 or Oct25 are boxed. The arrows denote supershifting.

FIGURE 4.

In vivo interaction of Oct25 to Gsc and Mix2 promoters. A, ChIP assays demonstrate that Gsc promoter regions containing either the Oct25 binding site (Gsc-A) or containing the DE and PE (Gsc-B) as well as the Mix2 promoter region spanning the ARE were amplified from chromatin precipitated by an Oct25 antibody. This amplification failed in the absence of antibody (no Ab). B, when Oct25 translation was blocked by Oct25MO, PCR products for Gsc and Mix2 promoter regions were reduced as compared with ctrlMO injections.

FIGURE 5.

Gsc promoter assays. A, diagram depicting the generation of Gsc promoter-luciferase reporter constructs. B, Oct25 represses Gsc -1500/+3 promoter activities stimulated by overexpression of Xnr1, Smad2, or by incubation with activin A protein. C and D, the same effect is observed for truncated promoters -479/+3(C) and -226/+3(D). E, VP16-XWBSCR11 alone or together with FAST1 stimulates an artificial reporter for DE, which is then repressed by Oct25.

FIGURE 6.

Mix2 promoter assays. A and B, Oct25 inhibits Mix2 -712/+13 promoter activity stimulated not only by overexpression of the ligand of nodal signaling, Xnr1 or Xnr5 (A), but also by overexpression of the transducers, Smad2 and/or FAST1 (B). C and D, Oct25 shows a repressive effect on the truncated Mix2 promoters -457/+13 (C) and -221/+13 (D), which are stimulated by Smad2 and/or FAST1.

FIGURE 7.

Analysis of Oct25 VP16 and EVE fusions on promoter activity and mesendoderm formation. A, wild-type Oct25 and VP16-Oct25 stimulate in contrast to EVE-Oct25 an artificial octamer reporter, suggesting that Oct25 is a transcriptional activator with regard to its octamer binding property. B, the controls demonstrate that stimulation is not due to the pGL3 vector, to which the octamer motifs were ligated. C and D, both Oct25 and VP16-Oct25 reveal repressive effect on Gsc (C) and Mix2 (D) promoters. E, stimulation of octamer reporter by VP16-Oct25 is inhibited by FAST1. F and G, either or revealed inhibition of Xbra (F) and Xsox17α (G), while in controls, the VP16 alone had no effect and EVE alone had weak effect on the expression of the two genes.

FIGURE 8.

Inhibition of HDAC activity did not alter the inhibitory effect of Oct25 on Gsc and Mix2. A, Gsc expression in wild-type gastrulae. B, circular expression of Gsc in TSA-treated embryos. C, dorsal injection of Oct25 mRNA leads to the disappearance of Gsc expression at the dorsal side in TSA-treated embryos. D and E, Mix2 showed similar expression in control (D) or TSA (E)-treated embryos. F, Oct25 overexpression leads to an inhibition of Mix2 expression in TSA-treated embryos.