Characterization of the sequences of the human cytomegalovirus enhancer that mediate differential regulation by natural and synthetic retinoids.

Abstract

Evidence exists to suggest that human cytomegalovirus (hCMV) may opportunistically use retinoic acid (RA) to advance its own replication, in which transcriptional activation of the viral major immediate-early promoter is a crucial control point. We demonstrate that the enhancer of the viral promoter contains three RA-response-elements that cooperate in mediating RA activation. These elements are direct repeats of two sequence motifs separated by 2 bp (DR2 site, REa) and 5 bp (DR5 sites, REb and c). DNA-binding experiments revealed that each of these elements bind RA receptor (RAR)-retinoid X receptor (RXR) heterodimers more efficiently than either homodimer. Apparent equilibrium dissociation constants of RAR-RXR heterodimers for sites REa, REb, and REc were estimated to be 5 nm, 10 nm, and 20 nm, respectively. The level of contribution of each of these elements to RA inducibility correlated with the strength of binding by RAR-RXR heterodimers to each site. These experiments demonstrate that RAR and RXR are necessary for RA responsiveness of the viral promoter. Using synthetic RA analogs, which selectively activate RARs and RXRs, the RAR partner within the heterodimeric complex appeared to be sufficient while the RXR partner was insufficient to independently activate transcription. However, joint activation of RARs and RXRs indicated that RXRs (in the presence of a transcriptionally active RAR) could contribute to transactivation. This restricted co-dependent ligand activation of RXR varied depending on the particular response element and the cell context. These studies further indicate that signaling of retinoid receptors (in particular RAR) by RA plays an important role in modulating hCMV infection.

Transcriptionally active drugs improve adenovirus vector performance in vitro and in vivo

“…CMV promoter retains repeated RARE and it is responsive to an in vitro RA treatment. 12 Based on our observations, the effect of RA may be the consenquence of a direct RXR/RAR activation and stimulation of CMV transcription. Further, TSA, increased three-to eight-fold in transgene production both in vitro and in vivo.…”

“…12 Typically, RA modulates gene expression by the activation of RARs that in turn associate with a number of co-activators some of which are members of the HAT family. In this context, the HDAC inhibitor TSA is known to enhance the transcriptional effect of RA.…”

“…The presence of multiple functional RARE elements in the CMV promoter sequence 12 prompted us to investigate whether the RA and TSA effects on transgene expression involve RARs activation. In transient transfection experiments endothelial cells were infected with 50 p.f.u.…”

“…[4][5][6] It retains cis regulatory elements recognized by ubiquitous transcription factors including AP-1, 7 CREB, 8 NF-kB 9,10 and retinoic acid nuclear receptors (RARs). 11,12 Despite these properties, CMV-based expression cassettes show timelimited transcriptional activity which is usually silenced within a few days after their introduction in target cells 3,10 both in vitro and in vivo. This phenomenon has been recently associated with the production of cytokines, such as interferon-␥ (INF-␥), capable of suppressing transgene expression.…”

Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells

“…Whether RXR is required to be liganded or functions as a`silent partner' of liganded RAR is still unclear. Several reports demonstrate RXR selective ligands fail to signal dierentiation in several tumor cell lines including NT2/D1 (Roy et al, 1995;Angulo et al, 1995;Minucci et al, 1997;Horn et al, 1996). However, recent studies using sub-optimal dosages of RAR and RXR selective ligands report synergistic induction of embryonal carcinoma dierentiation (Minucci et al, 1997;Roy et al, 1995;Horn et al, 1996).…”