Background: ONECUT transcription factors are characterized by the presence of a single CUT domain and a HOX domain. ONECUT2 is involved in pathogenesis of several solid tumors and regulates proliferation, migration and differentiation of tumor cells. We identified a transcript variant of ONECUT2 in a cDNA library of the chemo-resistant Hodgkin lymphoma (HL) cell line L-1236. This variant (ONECUT2s) contains a CUT Domain without a corresponding HOX domain. To further characterize this variant, we performed over-expression and knock-down studies in diverse cell line models and studied the impact of ONECUT2 variants on gene expression.
Methods: Expression of ONECUT2 and ONECUT2s in tissue samples and cell lines was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Additional expression studies were performed using public available microarray data sets. RNA interference was used for knock-down of ONECUT2 and ONECUT2s. For over-expression studies, the different ONECUT2 variants were cloned into the doxycycline-inducible vector pRTS-1, with a bi-directional promoter, allowing simultaneous expression of the transgene and the reporter EGFP. Knock-down and over-expression was analyzed by qRT-PCR. For assessing the influence of ONECUT2 and ONECUT2s on gene expression, microarray analysis and RNA-seq analyses were performed.
Results: Gene expression analysis by qRT-PCR showed high expression of ONECUT2 and ONECUT2s in HL-cell lines and normal liver. Public available microarray data also indicated high expression of ONECUT2 in HL-cell lines and a subset of HL biopsies. The majority of the other tissues and cell lines showed only low expression of both ONECUT2 variants. Microarray and RNA-seq analyses of transgenic cells and cells after knock-down of ONECUT2 variants showed that both variants affected the global gene expression profile. However, effects of ONECUT2 were much stronger than effects of ONECUT2s. Both variants seem to regulate different sets of genes, especially for ONECUT2 these genes include genes involved in apoptosis regulation and immune response.
Conclusion: ONECUT2 acts as a transcription factors in HL-cells and both variants have different effects on gene expression. The high expression of both ONECUT2 variants in HL might qualify this transcription factor as a possible candidate for targeted therapy of HL. Our work is supported by Mitteldeutsche Kinderkrebsforschung