Evaluation of a central core MDR region as an optimum chromatin opening model

Aim: To examine whether the sub-regions of the HNRPA2B1 CBX3 UCOE (A2UCOE) preserved UCOE functionality. This inquiry was driven by several reports that challenge the requirement of associated promoter activity for UCOE functionality. Method: The 0.9kb central A2UCOE region, thought to form a potential MDR (methylation determining region), was placed upstream of the SFFV-eGFP cassette. These constructs were subsequently analyzed alongside the 1.5A2UCOE and 2.2A2UCOE to assess their ability to inhibit transcriptional repression. Results: In this study, we investigated whether sub-regions of the HNRPA2B1 CBX3 UCOE (A2UCOE) retained UCOE activity. Lentiviral vectors were used to evaluate the stability and efficiency of eGFP expression in P19 and F9 embryonal carcinoma cells. LB medium was prepared, and recombinant plasmids were transformed into competent E. coli DH5α cells. HEK293T cells were cultured for lentiviral production and transduction experiments were performed. eGFP expression was analyzed by flow cytometry before and after differentiation of P19 and F9 cells. Statistical analysis was performed using Prism, with p < 0.05 considered significant. Conclusions: This study demonstrates that the 0.9 kb core region of A2UCOE, containing the promoters of HNRPA2B1 and CBX3, exhibits partial resistance to transgene silencing in both undifferentiated and differentiated P19 and F9 cells. The findings indicate that the size of the CpG-rich region is critical for maintaining open chromatin structure and ensuring full UCOE functionality. Our study highlights the potential of smaller A2UCOE subregions for gene therapy; however, further optimization is required to achieve full activity independent of promoter influence.