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. Human CBX2 exists in two isoforms, a 532-amino acid long isoform called CBX2.1, and a second shorter 211-aminoacid isoform named CBX2.2. The promoter of these variants are unknown, however there are hints of differential expression by the isoforms in different cell lines and tissues.
Objective and Hypothesis: 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, candidate regions targeting transcription and the start of translation, were cloned as reporter inserts into the pGL4.17 Vector which lacks a promoter, requires expression of SV40 T antigen, and encodes the luciferase reporter gene luc2. The custom promoter constructs were transfected in Cos-1 cells (SV40 transformed cell type), with reporter activity established by performing a dual-reporter assay measuring firefly and Renilla luciferases. Subsequently, CBX2 promoter elements are dissected based on predicted binding sites and expressed in ovarian, testicular and adrenal cell lines (KGN, NT2D-1, and NCI-H295R cells respectively) to determine the regulation of CBX2 expression.
Results: Utilizing the dual-reporter assay system, we identified an optimal candidate CBX2 promoter construct that exhibited a 3.6 normalized fold change in activity when compared to a negative control (P<0.0074). Preliminary results indicate that this promoter construct may be applied to investigate differential transactivation of CBX2 in cell models recapitulating ovaries, testis and adrenal cells.
Conclusion: The characterization of a candidate CBX2 promoter could elucidate the inner workings of a CBX2-regulated network and its functional role 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.
27 - 29 Sep 2018
European Society for Paediatric Endocrinology