The ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 housekeeping gene locus (A2UCOE) is able to provide highly reproducible and stable expression even from transgenes integrated within extreme heterochromatic regions (such as centromeres). The A2UCOE consists of the methylation-free CpG island encompassing the dual divergently transcribed promoters of HNRPA2B1 and CBX3. The proposed mechanism of A2UCOE function is a two-component model: (i) an extended methylation-free CpG island and (ii) dual divergent transcription with an inherent chromatin opening capability from the innate HNRPA2B1 and CBX3 promoters. Stable expression by the A2UCOE can be achieved from either driving transcription directly off the HNRPA2B1 promoter or by linkage to a silencing-prone heterologous ubiquitous or tissue specific promoter.
The A2UCOE has been shown to provide highly reproducible and stable transgene expression from within lentiviral vectors (LVs) both in vitro and more importantly in vivo following ex vivo gene transfer to mouse bone marrow haematopoietic stem cells (HSCs). It has recently been shown that efficient and stable expression of transgenes from the A2UCOE is at least in part due to its resistance to DNA methylation-mediated silencing.
The aim of this project was two-fold. First, to test the two-component model of A2UCOE function by evaluating other elements with a similar CpG island - dual divergent transcriptional promoter configuration. Second, to dissect the A2UCOE in an effort to identify sub-regions that are crucial for its dominant chromatin opening activity and thus obtain a minimal fully functional element. This would allow further savings of space for therapeutic gene sequences once incorporated within LVs.
Novel candidate UCOEs and A2UCOE sub-fragment-based eGFP reporter gene constructs within an LV context are functionally tested by transduction of P19 and F9 murine embryonic carcinoma cell lines and measurement of stability of expression over time both before and after differentiation down neuroectodermal and endodermal lineages respectively. Candidates showing encouraging signs of stability of expression in P19 and F9 cells would then also be tested for stability of expression in murine embryonic stem cells.
Results and Conclusions
The methylation-free CpG island, dual divergently transcribed regions used to test the overall two-component model of UCOE function were the native genomic SETD3-CCNK housekeeping gene pair and the artificially constructed single promoter RPS11-HNRPA2B1 combined in a divergent configuration. Linkage of these elements in both orientations upstream of the highly silencing prone SFFV-eGFP reporter gene system resulted in significant but only partial protection against silencing compared to the fully functional core 1.5A2UCOE-SFFV-eGFP reference. This was the case in P19 and F9 cell systems both before and after differentiation.
Various sub-fragments of the A2UCOE region ranging in size from 450-950bp either with or without associated promoter activity, were linked upstream of the SFFV-eGFP cassette and again compared to 1.5A2UCOE-SFFV-eGFP to assess their ability to negate transcriptional silencing. The results showed that none of the A2UCOE sub-fragments tested retained a full UCOE capability. Fragments from the CBX3 first intron lacking promoter activity, but with a high CpG content, were particularly noteworthy for their total inability to rescue expression from the silencing-prone SFFV promoter. A 0.9kb sub-core fragment of the 1.5kb A2UCOE extending over both CBX3 and HNRPA2B1 transcriptional start sites was only partially able to negate silencing of the linked SFFV promoter.
Analysis of a deletion series from the CBX3 end of a fully functional 2.2kb A2UCOE where expression of an eGFP reporter gene is directly driven off the HNRPA2B1 promoter, revealed a 1.7kb truncation that retained full UCOE activity following transduction of P19 and F9 cells in both undifferentiated and differentiated states. A further deletion to 1.2kb lacked this capability. This 1.7A2UCOE was also able to retain stable expression in murine embryonic stem cells and during differentiation into embryoid bodies.
In summary, we were able to identify a native (SETD3-CCNK) and construct an artificial (RPS11-HNRPA2B1) dual divergent promoter combination with a significant but partial UCOE function compared to the fully functional prototypical HNRPA2B1-CBX3 system. A2UCOE sub-fragments that were CpG rich but devoid of one or both of the CBX3 and HNRPA2B1 promoters, were at best only partially capable of conferring stability of expression. Lastly, we were able to substantially reduce the length of the A2UCOE required for full activity when gene expression is sought directly from the potent innate HNRPA2B1 promoter.
|Date of Award
|Michael Antoniou (Supervisor) & Gillian Bates (Supervisor)