Pou5f1/Oct4 Promotes Cell Survival via Direct Activation of mych Expression during Zebrafish Gastrulation

Abstract

Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of myc gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, myca, mycb, mych, mycl1a, mycl1b, and mycn, by whole mount in situ hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic pou5f1 mutant (MZspg) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage mych expression, as well as late gastrula stage mycl1b expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to mych and mycl1b control regions. The regulation of mych by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZspg embryos induced strong mych expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZspg embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed myc genes, or combination of several pathways affected in MZspg. Experimental mych overexpression in MZspg embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZspg gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZspg apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.

OCT4 expression regulated apoptosis and cell cycle in myeloma cells

“…Studies have also shown that overexpression of c-MYC through P53 results inhibition of proliferation and increased apoptosis (25). OCT4 plays an important role in c-MYC expression (26). Our findings also showed that changes in the cell cycle in the U266 cells line were not significant, but there were significant changes in the RPMI8226 cells in the G1 phase and S phase.…”

Functional interplay between MYCN, NCYM, and OCT4 promotes aggressiveness of human neuroblastomas

“…Previous reports have shown the physiological roles of OCT4 in the transcriptional regulation of MYC family members in various species. 24 – 26 OCT4 stimulates MYC transcription for cell proliferation in human and mouse ES cells 24 , 25 and activates myc transcription for cell survival during zebrafish gastrulation. 26 In the present study, we found the pathological significance of OCT4 for MYCN transcription in human neuroblastoma cells.…”

Maternal Nanog is required for zebrafish embryo architecture and for cell viability during gastrulation

“…Nanog and Oct4/Pou5f3 synergize with the ventral specification pathway via BMP signaling and Vox (Peres-Camps et al, 2016; this study). Pou5f3 regulates cell survival by inhibiting p53 (tp53) and activating mych (Kotkamp et al, 2014). The decrease in cell survival, observed in both Nanog and Oct4/Pou5f3 mutants, could be caused by multiple independent changes due to a widespread role of Nanog and Pou5f3 in gene expression.…”

“…We used a modified procedure from Kotkamp et al (2014). Embryos were fixed at 3, 4 and 4.7 hpf in Gard's FG fixative for 2 h and incubated overnight in PBST containing 0.33 M glycine.…”

“…In the maternal-zygotic (MZ) mutant of Pou5f3, MZspg (Spiel-ohne-Grenzen), two crucial components driving epiboly are severely affected: microtubules of the YCL and radial intercalation of deep cells (Reim and Brand, 2006;Lachnit et al, 2008;Song et al, 2013). Oct4/Pou5f3 mutants also show multiple independent patterning defects (Reim et al, 2004;Lunde et al, 2004;Belting et al, 2011), and increased apoptosis at the end of gastrulation (Kotkamp et al, 2014). In contrast, only epiboly movements are critically affected in MZeomesa mutants and in Mxtx2 knockdown (KD) embryos.…”