We isolated a 1-kb region of human genomic DNA directly upstream of the Artemis TSS by PCR amplification (APro). An in silico transcription factor binding site analysis of this sequence uncovered several transcription factor binding sites, leading us to further characterize the genomic region for promoter activity. 5′ RACE analysis indicated multiple transcriptional start points in RNA extracted from liver and lymphoid cell lines, and deletion analysis revealed divergent expression patterns between 293T cells and the BJAB Burkett lymphoma-like cell line. Transfection studies in vitro revealed the potential for this sequence to regulate gene expression in multiple cell types; additionally, APro mediated expression at a level that was substantially lower than that mediated by the strong EF1α promoter. Further, GFP expression regulated by APro was observed in vivo in mice transplanted with marrow that had been transduced with an APro-regulated lentiviral vector. Finally, expression was sustained in secondary transplant recipients in both myeloid and lymphoid lineages, establishing the effectiveness of APro to serve as a proficient promoter for gene expression within the hematopoeitic system.
The human APro region identified through this study exhibits several characteristics similar to those observed for promoters of other NHEJ proteins. The downstream promoter region extending from −184 to the human Artemis TSS contains neither a TATA box nor a CCAAT box, motifs also absent in the promoters for DNA-PKcs (Connelly et al
), Ku70 (Takiguchi et al
) and other housekeeping genes. Lack of a distinct TATA box within this region of the human APro sequence is associated with considerable variability in the location of transcriptional start sites, as determined by 5′ RACE, analogous to variability in transcriptional initiation sites observed for other NHEJ messages such as DNA-PKcs, in which at least 6 transcriptional start sites were observed (Connelly et al
). We also tested full-length APro and deletion constructs transfected into 293T cells for the effect of irradiation or exposure to the radiomimetic chemicals bleomycin and etoposide, but induced expression upon DNA damage was not observed (data not shown). Similarly, neither transcription nor translation of DNA-PKcs are induced upon DNA damage in either human or mouse cells (Lee et al
; Connelly et al
). These observations indicate the similarity of human Artemis transcriptional regulation with that of other NHEJ components.
Our 5′ RACE results and the in vitro
deletion mapping presented in this study suggest the potential for multiple regulatory regions that comprise the endogenous Artemis promoter. This is consistent with Artemis' involvement in several distinct cellular functions such as NHEJ (Ma et al
), V(D)J recombination (Ma et al
), and apoptosis (Britton et al
). It is tempting to speculate that Artemis expression may be spatially and/or temporally regulated per required function, similar to several other proteins demonstrated to be differentially regulated through transcript initiation at multiple promoter sites. For example, the murine α-amylase gene exhibits tissue specific expression regulated by two separate promoters (Schibler et al
). S1 nuclease mapping revealed that liver tissues yielded one minor α-amylase transcript, whereas the parotid gland yielded two α-amylase transcripts including the minor transcript plus one additional major transcript; additionally, synthesis of each transcript was found to initiate directly downstream of TATA boxes identified by sequence analysis (Schibler et al
). Our data show two potential promoter regions regulating human Artemis expression: one containing a well-defined TATA box with the ability to regulate expression in B-lymphoid cells and the downstream promoter region being less defined in that it does not contain a TATA box, yet regulates expression in several other cell types. It will be of interest to study the relationship between Artemis function and tissue type expression and address the possibility that one region of the APro sequence is necessary for expression during V(D)J recombination, whereas the other region confers constitutive expression in support of NHEJ.
Gene transfer is emerging as a promising approach for treatment of genetic disorders. However, recently observed adverse events have called to attention the importance of regulating expression of therapeutic genes. Specifically, two independent studies have reported correction of X-linked SCID by ex vivo
transduction of CD34+
hematopoeitic stem cells using a retroviral vector expressing the common cytokine-receptor gamma chain (common γ chain) (Gaspar et al
; Hacein-Bey-Abina et al
). Long-term engraftment of corrected stem cells was observed in the majority of patients, ultimately resulting in reconstitution of a functional lymphocyte compartment. To date, however, five out of twenty patients treated for X-linked SCID by gene transfer have developed T cell outgrowth resulting in leukemia, from which one child has died (Howe et al
; Hacein-Bey-Abina et al
). Although insertional activation of the LMO2
oncogene was reported in four of the leukemic cases, overexpression of the common γ chain induces cellular proliferation and thus may have contributed to the T lymphocyte clonal outgrowth (Howe et al
; Amorosi et al
). More tightly regulated expression of the common γ chain may thus reduce the risk of oncogenesis resulting from common γ chain overexpression.
Achieving transgenic expression of human Artemis for the correction of SCID-A will be challenging considering previous results regarding Artemis overexpression. Recently, two independent groups reported correction of a murine model of SCID-A by lentiviral vector-mediated gene transfer (Mostoslavsky et al
; Benjelloun et al
). In both studies, SCID-A animals receiving HSC transduced with a lentiviral vector encoding a human Artemis cDNA regulated by the PGK promoter exhibited repopulation of both B and T lymphocyte compartments. However, Mostoslavsky et al
. reported the inability of either CMV or EF1α regulated human Artemis lentiviral vectors to restore B and T cells in RAG-1 deficient animals receiving SCID-A HSCs transduced with either vector (Mostoslavsky et al
; Benjelloun et al
). Considering the endonucleolytic nature of Artemis, these results lead us to consider whether Artemis overexpression might be inherently toxic. We subsequently characterized the effect of Artemis overexpression and found it to be associated with cytotoxicity, ultimately resulting in a halt in cell cycle progression, fragmentation of genomic DNA, and apoptosis (Multhaup et al
). These results emphasize the importance of providing Artemis expression at a level that is nontoxic and yet sufficient to correct the T−
phenotype in preclinical studies and in clinical application to human SCID-A.
The endogenous human Artemis promoter provides expression both in vitro and in vivo at levels significantly lower than those of well-characterized strong promoters such as CMV and EF1α. Further, when studied in vivo, the Artemis promoter regulated expression in several lymphoid cell populations, demonstrating the ability of Artemis promoter to mediate expression in important hematopoeitic compartments after gene transfer into hematopoietic stem cells. The APro sequence thus has great potential as a regulator of therapeutic gene expression, including expression of the human Artemis gene for in vivo correction of human SCID-A. With this in mind, we have recently generated lentiviral vectors employing innate regulation of human Artemis cDNA using its own endogenous promoter sequence and have achieved successful ex vivo gene transfer resulting in functional lymphocyte repopulation of a murine model of SCID-A (M. Multhaup et al., manuscript in preparation). Overall, these results demonstrate that providing innate Artemis expression via ex vivo lentiviral transduction into hematopoietic stem cells may serve as a clinically relevant and feasible treatment of human SCID-A. As an element that supports moderate but reliable levels of expression, the human APro also has the potential for reduced genotoxicity associated with integration of other therapeutic genes.