ESPE Abstracts

Background: The process of sexual differentiation is critical for reproduction in nearly all metazoan. Defects in any of the genes involved in either testicular or ovarian development can result in disorders of sex development (DSD). CBX2/M33 is a chromatin modifier that plays an important role in sexual development and its disorders, highlighted by the fact that M33-deficient mice have male-to-female sex reversal and loss-of-function of CBX2 causes 46, XY DSD in humans. The promoter of CBX2 is unknown; however, there are hints of differential expression by its multiple isoforms across different cell lines and tissues.

Here we aim to characterize the CBX2 promoter in applicable cell lines using a custom reporter construct, to identify a regulatory network in gonadal development in which CBX2 takes part.

Methods: To locate the CBX2 promoter, based on predicted binding sites two candidate regions targeting transcription and one the start of translation, were cloned into the promoterless pGL4.17 Vector upstream of the luciferase reporter gene luc2. The constructs were transfected in several cell lines (Cos-1, HeLa, KGN, and NT2-D1), with reporter activity established by performing a dual-reporter assay measuring firefly and Renilla luciferases. Dissection of the CBX2 promoter is done to determine essential transcription factor binding sites and investigate DNase I hypersensitive sites, along with related histone activity. Subsequently, ovarian, testicular and adrenal cell lines (KGN, NT2D-1, and NCI-H295R cells respectively) are challenged with the identified regulatory elements to determine the regulation of CBX2 expression.

Results: Utilizing the dual-reporter assay system, we identified an optimal candidate CBX2 promoter construct (-479/+34) that exhibited a significant normalized fold change in activity across several cell lines tested (range from 3.6 – 14.65 fold) when compared to a negative control (P<0.0074 – P<0.0001). Preliminary results indicate substantial differences in transactivation potential among the various cells, allowing us the potential application of the promoter construct to explore and elucidate differential transactivation of CBX2 in cell models recapitulating ovaries, testis and adrenal cells.

Conclusion: The characterization of a candidate CBX2 promoter could provide insight into the functional role of CBX2 as transactivator, distinct from its known function as chromatin-modifier. Further study of the impact of CBX2 activation and suppression may shed light on potential pathological mechanisms involved in DSD, and ultimately its diagnosis and management.