Dataset Information

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

ABSTRACT: Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF(-) (/) (-) ) ESC, we discovered that loss of GCNF leads to the repression of cyclin D1 activation during ESC differentiation. This was determined to be an indirect effect of deregulation Mir302a, which is a cyclin D1 suppressor via binding to the 3'UTR of cyclin D1 mRNA. Moreover, we showed that Mir302 is a target gene of GCNF that inhibits Mir302 expression by binding to a DR0 element within its promoter. Inhibition of Mir302a using Mir302 inhibitor during differentiation of GCNF(-) (/) (-) ESCs restored cyclin D1 expression. Similarly over-expression of GCNF during differentiation of GCNF(-) (/) (-) ESCs rescued the inhibition of Mir302a expression and the activation of cyclin D1. These results reveal that GCNF plays a key role in regulating activation of cyclin D1 expression via inhibition of Mir302a.

SUBMITTER: Wang H

PROVIDER: S-EPMC4091926 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

Wang Hongran H Wang Xiaohong X Archer Trevor K TK Zwaka Thomas P TP Cooney Austin J AJ

Stem cells (Dayton, Ohio) 20140601 6

Cyclin D1 plays an important role in the regulation of cellular proliferation and its expression is activated during gastrulation in the mouse; however, it remains unknown how cyclin D1 expression is regulated during early embryonic development. Here, we define the role of germ cell nuclear factor (GCNF) in the activation of cyclin D1 expression during embryonic stem cell (ESC) differentiation as a model of early development. During our study of GCNF knockout (GCNF(-) (/) (-) ) ESC, we discovere ...[more]

PMID: 24578347

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Project description:p107 functions to control cell division and development through interaction with members of the E2F family of transcription factors. p107 is phosphorylated in a cell cycle-regulated manner, and its phosphorylation leads to its release from E2F. Although it is known that p107 physically associates with E- and A-type cyclin/cyclin-dependent kinase 2 (Cdk2) complexes through a cyclin-binding RXL motif located in the spacer domain, the mechanisms underlying p107 inactivation via phosphorylation remain poorly defined. Recent genetic evidence indicates a requirement for cyclin D1/Cdk4 complexes in p107 inactivation. In this work, we provide direct biochemical evidence for the involvement of cyclin D1/Cdk4 in the inactivation of p107's growth-suppressive function. While coexpression of cyclin D1/Cdk4 can reverse the cell cycle arrest properties of p107 in Saos-2 cells, we find that p107 in which the Lys-Arg-Arg-Leu sequence of the RXL motif is replaced by four alanine residues is largely refractory to inactivation by cyclin D/Cdk4, indicating a role for this motif in p107 inactivation without a requirement for its tight interaction with cyclin D1/Cdk4. We identified four phosphorylation sites in p107 (Thr-369, Ser-640, Ser-964, and Ser-975) that are efficiently phosphorylated by Cdk4 but not by Cdk2 in vitro and are also phosphorylated in tissue culture cells. Growth suppression by p107 containing nonphosphorylatable residues in these four sites is not reversed by coexpression of cyclin D1/Cdk4. In model p107 spacer region peptides, phosphorylation of S640 by cyclin D1/Cdk4 is strictly dependent upon an intact RXL motif, but phosphorylation of this site in the absence of an RXL motif can be partially restored by replacement of S643 by arginine. This suggests that one role for the RXL motif is to facilitate phosphorylation of nonconsensus Cdk substrates. Taken together, these data indicate that p107 is inactivated by cyclin D1/Cdk4 via direct phosphorylation and that the RXL motif of p107 plays a role in its inactivation by Cdk4 in the absence of stable binding.

Dataset Information

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

Publications

GCNF-dependent activation of cyclin D1 expression via repression of Mir302a during ESC differentiation.

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