On the role of chromatin in the regulation of adenovirus vector transgene expression

Abstract

Adenovirus vectors are undergoing evaluation in clinical trials for the treatment of a variety of human diseases. However, little is clearly understood regarding interactions between non-replicating adenovirus (Ad) vectors and the host nucleus. Here I have examined the role of chromatin in the regulation of helper-dependent Ad (hdAd) vector-encoded transgenes. Eviction of the Ad DNA packaging-protein VII, histone deposition, and transgene expression initiated within 2 hours of infection; hdAd assembled into physiologically-spaced nucleosomes within 6 hours. Histone deposition was transcription-independent, dependent on the histone chaperone HIRA, and essential for efficient viral gene expression. Having established that hdAd vectors assemble into chromatin, I examined the role of epigenetic factors in mediating the repressive effects of prokaryotic DNA attached to eukaryotic transgenes. To this end, I used helper-dependent adenovirus (hdAd) vectors with identical expression cassettes, but containing 22 kb of prokaryotic or eukaryotic stuffer DNA (hdAd-prok and hdAd-euk, respectively). I hypothesized that abundant CpG motifs in the hdAd-prok backbone are methylated, resulting in the formation of chromatin that is refractory to transcription. Within the host cell, the hdAd-prok transgene promoter was not associated with epigenetic markers of heterochromatin, although it did exhibit reduced association with acetylated histones H3 and H4. Inhibition of histone deacetylases or insertion of an insulator element between the transgene and stuffer DNA abrogated the inhibitory effects of prokaryotic DNA. However, I did not detect methylation of hdAd-prok DNA. Therefore, I examined the role of promyelocytic leukemia (PML) bodies and their constituents, Sp100 and Daxx - which have all been implicated in repression of DNA virus transcription - in repression of hdAd-prok. Intact PML bodies and the PML protein were not necessary for repression of hdAd-prok. Rather, Sp100 isoforms B and HMG, which bind to unmethylated CpGs, and Daxx, which interacts with histone deacetylases, repressed expression of hdAd-prok. Therefore, "foreign" prokaryotic DNA is recognized by Sp100B/HMG and Daxx, which repress associated genes via induction of histone deacetylation. I found that chromatin plays an important role in the regulation of Ad vector-encoded transgenes, suggesting that both genetic and epigenetic factors must be considered in the design of Ad vectors for gene therapy.