Role of CBX2 isoforms in human sex development

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 586-595-Reproductive Axis Determination, Development & Transgender Medicine
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-587
Mariangela Franco*1, Berenice Bilharinho Mendonca2, Elaine Maria Frade Costa3 and Anna Lauber-Biason4
1university of Fribourg, Fribourg, Switzerland, 2Univ Sao Paulo Fac Med, Sao Paulo, Brazil, 3Hospital das Clinicas, Faculdade de Medicina, Universidade de São Paulo, Sao Paulo, Brazil, 4University of Fribourg, Fribourg, Switzerland
CBX2/M33 is a regulator of chromatin structure and chromosome architecture at its target loci.

Two splicing variants of CBX2 are known: CBX2.1 and CBX2.2 share the first three exons, but the variant 2 is much shorter than the variant 1.

CBX2.1. Recently, our group identified CBX2.1 as an essential transactivator for human male gonadal development. Overexpression of CBX2.1 in NT2-D1 Sertoli-like cells stimulates SOX9 and SF1endogenous expression (3.5 and 6 fold respectively). Transactivation studies based on enriched ChIP fragments suggested that CBX2.1 stimulates SOX9 expression directly in the testis whereas it limits SOX9 expression in the ovary. To understand the exact position of CBX2.1 in sex development cascade, we investigated the role of CBX2.1 as transcription factor on SOX9 promoter. Different constructs of the SOX9 promoter were cloned upstream of a firefly luciferase reporter vector and transactivation studies were performed in NT2D1 cells.

Our data showed that exogenous CBX2.1 in NT2D1 cells did not significantly change luciferase activity driven by any of the SOX9 promoter constructs. These same constructs were active and stimulatory when transfected into the mouse mesenchymal C3H10T1/2 cells, demonstrating that our results are due to the different biology of the cell lines and not to an experimental artifact.

CBX2.2. A mutation in CBX2.2 (p.C132R) has been identified in a 46,XY patient with complete female phenotype and fibrotic testes. “In silico” modeling of CBX2.2 protein strongly indicated a deleterious effect.To test the functional consequences of this mutation, we performed transactivation studies to investigate the potential role of CBX2.2 on SF1 promoter, using different constructs upstream of a firefly luciferase reporter as a putative target for CBX2.2 in H295R adrenal-corticocarcinoma cells. Transfection of CBX2.2 in these cells had no effect on any tested construct.

To summarize, CBX2.1 increases SOX9 and SF-1 expression in NT2D1 Sertoli-like cells. This effect is not directly mediated by their promoter sequences. The role of CBX2.2 in human sex development remains unclear.

Given the well-known importance of the testis-specific enhancer (hTES) in the regulation of SOX9 expression, we are presently studying the effect of both CBX2 isoforms on this regulatory element. In parallel, experiments using small hairpin RNA (shRNA) to stably knock-down of both CBX2 in NT2D1 cells will allow us to establish their effect on the expression of the endogenous SOX9 and other genes involved in sex development.

Nothing to Disclose: MF, BBM, EMFC, AL

*Please take note of The Endocrine Society's News Embargo Policy at

Sources of Research Support: Swiss National Science Foundation Grant 320030_130645