Institut de
Génomique
Fonctionnelle

We originally got interested in genomic imprinting during the functional characterization of a zinc finger transcription factor that we named Zac1. We showed that this transcription factor is able to induce apoptosis and cell cycle arrest in different cell types (Spengler et al. 1997 ; Varrault et al. 1998 ; Bilanges et al. 2001), suggesting a possible role as a tumor suppressor gene (Bilanges et al. 1999 ; Basyuk et al. 2005).

Figure 1 : Epithelial LLC-PK1 cells were transfected by the Tet-off system that allows regulated Zac1 expression. In the presence of tetracycline (left panel), Zac1 is not expressed whereas in the absence of the antibiotic (right panel) Zac1 is induced (green label). Cortical actin was labeled by phalloidine (red label). Somme of the cells expressing Zac1 display a fragmented nucleus indicative of apoptosis.

More recently, ZAC was shown to be maternally imprinted in humans (Kamiya et al. 2000) and mice (Piras et al. 2000). ZAC is located in the 450 kb minimal interval for Transient Neonatal Diabetes Mellitus (TNDM) (Gardner et al. 2000), a rare genetic disease due to the overexpression of a maternally imprinted gene that affect the maturation and/or differentiation of pancreatic beta-cells. Because of its functional properties, its chromosomal localization, its maternal imprinting, and the regulation of its promoter (Varrault et al. 2001), ZAC is the TNDM candidate gene. We presently look for Zac1 target genes, and want to understand the mechanims that underly Zac1 extinction in some tumors, its mechanisms of action, and its role during embryonic development.

Figure 2 : Zac1 is widely expressed during mouse embryonic development.

We generated a Zac1 nullizygote mouse mutant, which, as is the case for a number of other imprinted genes, displays alterations of embryonic growth (Varrault et al. 2006). Using the results of a meta-analysis of microarray data, we showed that Zac1 belongs to a gene network that comprises other imprinted genes and controls embryonic growth (Varrault et al. 2006).

Figure 3 : The Imprinted Gene Network (IGN). A network of co-regulated genes was discovered using the results of a meta-analysis of 116 microarray data sets. Imprinted genes are in bold. The edge thickness is proportional to the number of experiments in which these 2 genes are co-regulated. Details regarding the construction of this network are found in Varrault et al. (2006) Dev. Cell 11:711-22.

We presently better characterize this network, and seek for the bioological process its controls and the mechanisms that ensure the cooridinated regulation of this set of genes. For that purpose, we use an in vitro model of corticogenesis that allows to generate every cortical neuron subtype from embryonic stem cells (Gaspard et al. 2008, 2009).

Figure 4 : Embryonic stem cells were induced to differentiate in cortical neurons (green) and glial cells (red) for 21 days. Cell nuclei were counterstained with DAPI (blue).

Références :

• Basyuk E, Coulon V, Le Digarcher A, Coisy-Quivy M, Moles JP, Gandarillas A, Journot L (2005) The candidate tumor suppressor gene ZAC is involved in keratinocyte differentiation, and its expression is lost in basal cell carcinomas. Mol Cancer Res 3:483-92
• Bilanges B, Varrault A, Basyuk E, Rodriguez C, Mazumdar A, Pantaloni C, Bockaert J, Theillet C, Spengler D, Journot L (1999) Loss of expression of the candidate tumor suppressor gene ZAC in breast cancer cell lines and primary tumors. Oncogene 18 : 3979-88
• Bilanges B, Varrault A, Mazumdar A, Pantaloni C, Hoffmann A, Bockaert J, Spengler D, Journot L (2001) Alternative splicing of the imprinted candidate tumor suppressor gene ZAC regulates its antiproliferative and DNA binding activities. Oncogene 20 : 1246-53
• Gardner RJ, Mackay DJ, Mungall AJ, Polychronakos C, Siebert R, Shield JP, Temple IK, Robinson DO (2000) An imprinted locus associated with transient neonatal diabetes mellitus. Hum Mol Genet 9:589-96
• Gaspard N, Bouschet T, Hourez R, Dimidschstein J, Naeije G, van den Ameele J, Espuny-Camacho I, Herpoel A, Passante L, Schiffmann SN, Gaillard A, Vanderhaeghen P (2008) An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 455:351-7
• Gaspard N, Bouschet T, Herpoel A, Naeije G, van den Ameele J, Vanderhaeghen P (2009) Generation of cortical neurons from mouse embryonic stem cells. Nat Protoc 4:1454-63
• Kamiya M, Judson H, Okazaki Y, Kusakabe M, Muramatsu M, Takada S, Takagi N, Arima T, Wake N, Kamimura K, Satomura K, Hermann R, Bonthron DT, Hayashizaki Y (2000) The cell cycle control gene ZAC/PLAGL1 is imprinted—a strong candidate gene for transient neonatal diabetes. Hum Mol Genet 9:453-60
• Piras G, El Kharroubi A, Kozlov S, Escalante-Alcalde D, Hernandez L, Copeland NG, Gilbert DJ, Jenkins NA, Stewart CL (2000) Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon-sarcoglycan are maternally imprinted genes : identification by a subtractive screen of novel uniparental fibroblast lines. Mol Cell Biol 20:3308-15
• Spengler D, Villalba M, Hoffmann A, Pantaloni C, Houssami S, Bockaert J, Journot L (1997) Regulation of apoptosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain. EMBO J. 16 : 2814-25
• Varrault A, Ciani E, Apiou F, Bilanges B, Hoffmann A, Pantaloni C, Bockaert J, Spengler D, Journot L (1998) hZAC encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer. Proc Natl Acad Sci USA 95 : 8835-40
• Varrault A, Bilanges B, Mackay DJG, Basyuk E, Ahr B, Fernandez C, Robinson DO, Bockaert J, Journot L (2001) Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus. J Biol Chem 276 : 18653-6
• Varrault A, Gueydan C, Delalbre A, Bellmann A, Houssami S, Aknin C, Severac D, Chotard L, Kahli M, Le Digarcher A, Pavlidis P, Journot L (2006) Zac1 regulates an imprinted gene network critically involved in the control of embryonic growth. Dev Cell 11:711-22