A 10-base-pair element of the human immunodeficiency virus type 1 long terminal repeat (LTR) is an absolute requirement for transactivation by the human cytomegalovirus 72-kilodalton IE1 protein but can be compensated for by other LTR regions in transactivation by the 80-kilodalton IE2 protein.

Transient gene expression studies have indicated that human cytomegalovirus (HCMV) specifically transactivates the human immunodeficiency virus (HIV) long terminal repeat (LTR). We show here, by a specific mutational analysis, that only the TATA box region is obligatory for transactivation of the HIV-1 LTR by HCMV. Similarly, this element is also sufficient for transactivation by either the HCMV 72-kDa major immediate-early 1 (IE1) or 80-kDa IE2 gene product independently. However, deletion of a 10-bp region from the minimal responsive element, 5' to the TATA box, dramatically reduced the level of HCMV 72-kDa IE1 or 80-kDa IE2 transactivation, indicating a crucial role for this element in transactivation. Whereas inclusion of the TAR element or Sp1 sites on this 10-bp-deleted minimal promoter had no effect on the removal of IE1 transactivation, TAR and Sp1 elements did compensate for the 10-bp element in transactivation by IE2 and HCMV. Consequently, the sequence requirements of the HIV-1 LTR for transactivation by HCMV can be reproduced by these IE1 and IE2 gene products of HCMV.     

Artificial recruitment of Sp1 or TBP can replace the role of IE1 in the synergistic transactivation by IE1 and IE2

The IE1 and IE2 proteins of human cytomegalovirus transactivate various viral and cellular promoters in a synergistic manner, but the mechanism of their action has not been well elucidated. Here we have examined the IE1-IE2 synergy by artificial recruitment of either Sp1 or TBP to the promoter. We found that in the presence of Sp1, the synergistic effect of IE1 on IE2-mediated transactivation dramatically decreased. Furthermore, a 117-amino acids glutamine-rich fragment of Sp1, which can interact with dTAF(II)110 and hTAF(II)130, was sufficient to replace the role of IE1 in IE1-IE2 synergism. It was also found that TBP recruitment to the promoter markedly decreased the synergistic effect of IE1 on IE2-mediated transactivation. These results suggested that in the context of the synergism between IE1 and IE2, the function of IE1 might overlap with that of Sp1, for example by recruiting the TFIID complex.     

Varicella-zoster virus IE62 protein utilizes the human mediator complex in promoter activation

The varicella-zoster virus (VZV) major transactivator, IE62, is involved in the expression of all kinetic classes of VZV genes and can also activate cellular promoters, promoters from heterologous viruses, and artificial promoters containing only TATA elements. A key component of the mechanism of IE62 transactivation is an acidic activation domain comprising the N-terminal 86 amino acids of IE62. However, the cellular target of this N-terminal acidic activation is unknown. In the work presented here, we show that the IE62 activation domain targets the human Mediator complex via the Med25 (ARC92) subunit and that this interaction appears to be fundamental for transactivation by the IE62 activation domain. In contrast, the Med23 subunit (Sur2/TRAP150beta/DRIP130/CRSP130) of the Mediator complex is not essential for IE62-mediated activation. Further, the IE62 activation domain appears to selectively interact with a form of the Mediator complex lacking CDK8. Chromatin immunoprecipitation experiments showed that IE62 stimulates recruitment of Mediator to an IE62-responsive model promoter. Finally, immunofluorescence microscopy of VZV-infected cells demonstrated intranuclear translocation of the Mediator complex to viral replication compartments. These studies suggest that Mediator is an essential component for efficient VZV gene expression.     

Molecular mechanisms that distinguish TFIID housekeeping from regulatable SAGA promoters

An important distinction is frequently made between constitutively expressed housekeeping genes versus regulated genes. Although generally characterized by different DNA elements, chromatin architecture and cofactors, it is not known to what degree promoter classes strictly follow regulatability rules and which molecular mechanisms dictate such differences. We show that SAGA‐dominated/TATA‐box promoters are more responsive to changes in the amount of activator, even compared to TFIID/TATA‐like promoters that depend on the same activator Hsf1. Regulatability is therefore an inherent property of promoter class. Further analyses show that SAGA/TATA‐box promoters are more dynamic because TATA‐binding protein recruitment through SAGA is susceptible to removal by Mot1. In addition, the nucleosome configuration upon activator depletion shifts on SAGA/TATA‐box promoters and seems less amenable to preinitiation complex formation. The results explain the fundamental difference between housekeeping and regulatable genes, revealing an additional facet of combinatorial control: an activator can elicit a different response dependent on core promoter class.