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RING finger protein 17 (Rnf17) of Myc/Max/Mxd network: expression and function during mouse reproduction and development

RING finger protein 17 (Rnf17) of Myc/Max/Mxd network: expression and function during mouse reproduction and development
RING finger protein 17 (Rnf17) of Myc/Max/Mxd network: expression and function during mouse reproduction and development
The preimplantation mammalian embryo is sensitive to the environment in which they develop and grow, either in vivo or in vitro. Disturbance to embryo environmental conditions can affect embryo growth. Changes in gene expression is one of the embryo responses to such conditions that may influence developmental potential and phenotype during later gestation. The signalling network Myc/Max/Mxd function as a molecular switch that regulates cell growth, proliferation and differentiation by controlling a common set of genes. RING finger protein 17(Rnf17) enhance Myc activity by sequestering all four members of Mxd family and creating a "Mxd-null phenotype". The MXD family functions as MYC repressor proteins. Two isoforms were described for Rnf17, long (Rnf17L) and short (Rnf17S). Rnf17 is expressed in adult mouse testis and in the mouse preimplantation embryo. The Rnf17 proteins contain RING finger domain and varying copies of the Tudor repeat domain (Tdrd). The aim of my thesis was to study the potential for Rnf17 regulating the Myc/Max/Mxd network during preimplantation development on the phenotype of the blastocyst and foetal testes. First, F9 murine carcinoma cells were used to question whether Rnf17 can modulate Myc responses. Functional assays were developed for Rnf17 over-expression by expression plasmids or Rnf17 knockdown by RNAi construct. The specificity of Rnf17 E3 ligase was investigated by co-transfection of Flagg-tagged constructs for RNF17 and MXD1 in F9 cells. Second, expression of Rnf17 mRNA and subcellular localisation of RNF17 protein were examined in mouse preimplantation embryo and in E17.5 foetal testes under normal condition and in response to maternal protein diets (LPD; 9% casein, HPD; 30% casein and control NPD; 18% casein). Finally Rnf17 knockdown was performed in mouse embryo by siRNA microinjection to point out the role of Rnf17 in embryo development.

RNF17 was able to degrade MXD1 by ubiquitinylation pathway in co-transfected Cos-1 cells. Rnf17-SV40 expression construct significantly induced Luciferase activity of c-Myc reporter construct (P?0.05) when over-expressed in F9 cells, and knockdown of Rnf17 using Rnf17-RNAi construct significantly reduced Luciferase activity of Rnf17-sensor construct (P?0.05). Rnf17 mRNA is expressed through preimplantation development, in E17.5 foetal testes and in F9 cells. Members of Myc/Max/Mxd are expressed in mouse preimplantation embryo. The expression pattern of RNF17 in F9 cells and mouse embryos was predominantly nuclear and also presented in the cytoplasm. RNF17 was only expressed in the nuclei of Sertoli cells with faint signal in prospermatogonia in E17.5 foetal testes. Knockdown of Rnf17 in microinjected embryos using Rnf17_siRNA reduced expression of Rnf17S, delayed blastocyst formation, and reduced RNF17 protein signal. Maternal diet differentially regulate expression of Rnf17 and c-Myc in mouse blastocyst and E17.5 testes.

In conclusion, over-expression of Rnf17 can amplify Myc response in F9 cells. MXD is a target protein for Rnf17 E3 ligase. Knockdown of Rnf17 in mouse embryo delayed blastocyst development. This study implicates that Rnf17 involved in regulating Myc/Max/Mxd signalling network and indicates a role for Rnf17 in blastocyst proliferation and growth. Changes in Rnf17 expression have the capacity to modulate early embryonic development and may contribute to phenotypic changes occurring later in life.
Bakheet, Ayat
568e5a14-48f3-4c3c-b89f-06d4af504c65
Bakheet, Ayat
568e5a14-48f3-4c3c-b89f-06d4af504c65
Fleming, Thomas
2abf761a-e5a1-4fa7-a2c8-12e32d5d4c03
Barton, Charles
5dfb4e1a-d559-4b58-9036-31de1e6c9ad8

(2013) RING finger protein 17 (Rnf17) of Myc/Max/Mxd network: expression and function during mouse reproduction and development. University of Southampton, Biological Sciences, Doctoral Thesis, 276pp.

Record type: Thesis (Doctoral)

Abstract

The preimplantation mammalian embryo is sensitive to the environment in which they develop and grow, either in vivo or in vitro. Disturbance to embryo environmental conditions can affect embryo growth. Changes in gene expression is one of the embryo responses to such conditions that may influence developmental potential and phenotype during later gestation. The signalling network Myc/Max/Mxd function as a molecular switch that regulates cell growth, proliferation and differentiation by controlling a common set of genes. RING finger protein 17(Rnf17) enhance Myc activity by sequestering all four members of Mxd family and creating a "Mxd-null phenotype". The MXD family functions as MYC repressor proteins. Two isoforms were described for Rnf17, long (Rnf17L) and short (Rnf17S). Rnf17 is expressed in adult mouse testis and in the mouse preimplantation embryo. The Rnf17 proteins contain RING finger domain and varying copies of the Tudor repeat domain (Tdrd). The aim of my thesis was to study the potential for Rnf17 regulating the Myc/Max/Mxd network during preimplantation development on the phenotype of the blastocyst and foetal testes. First, F9 murine carcinoma cells were used to question whether Rnf17 can modulate Myc responses. Functional assays were developed for Rnf17 over-expression by expression plasmids or Rnf17 knockdown by RNAi construct. The specificity of Rnf17 E3 ligase was investigated by co-transfection of Flagg-tagged constructs for RNF17 and MXD1 in F9 cells. Second, expression of Rnf17 mRNA and subcellular localisation of RNF17 protein were examined in mouse preimplantation embryo and in E17.5 foetal testes under normal condition and in response to maternal protein diets (LPD; 9% casein, HPD; 30% casein and control NPD; 18% casein). Finally Rnf17 knockdown was performed in mouse embryo by siRNA microinjection to point out the role of Rnf17 in embryo development.

RNF17 was able to degrade MXD1 by ubiquitinylation pathway in co-transfected Cos-1 cells. Rnf17-SV40 expression construct significantly induced Luciferase activity of c-Myc reporter construct (P?0.05) when over-expressed in F9 cells, and knockdown of Rnf17 using Rnf17-RNAi construct significantly reduced Luciferase activity of Rnf17-sensor construct (P?0.05). Rnf17 mRNA is expressed through preimplantation development, in E17.5 foetal testes and in F9 cells. Members of Myc/Max/Mxd are expressed in mouse preimplantation embryo. The expression pattern of RNF17 in F9 cells and mouse embryos was predominantly nuclear and also presented in the cytoplasm. RNF17 was only expressed in the nuclei of Sertoli cells with faint signal in prospermatogonia in E17.5 foetal testes. Knockdown of Rnf17 in microinjected embryos using Rnf17_siRNA reduced expression of Rnf17S, delayed blastocyst formation, and reduced RNF17 protein signal. Maternal diet differentially regulate expression of Rnf17 and c-Myc in mouse blastocyst and E17.5 testes.

In conclusion, over-expression of Rnf17 can amplify Myc response in F9 cells. MXD is a target protein for Rnf17 E3 ligase. Knockdown of Rnf17 in mouse embryo delayed blastocyst development. This study implicates that Rnf17 involved in regulating Myc/Max/Mxd signalling network and indicates a role for Rnf17 in blastocyst proliferation and growth. Changes in Rnf17 expression have the capacity to modulate early embryonic development and may contribute to phenotypic changes occurring later in life.

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Published date: 30 September 2013
Organisations: University of Southampton, Centre for Biological Sciences

Identifiers

Local EPrints ID: 367078
URI: http://eprints.soton.ac.uk/id/eprint/367078
PURE UUID: f7cc571d-40cc-43a4-97d9-7c5acf502674

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Date deposited: 22 Oct 2014 12:48
Last modified: 18 Jul 2017 02:04

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Contributors

Author: Ayat Bakheet
Thesis advisor: Thomas Fleming
Thesis advisor: Charles Barton

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