Gene target specificity of the Super Elongation Complex (SEC) family: how HIV-1 Tat employs selected SEC members to activate viral transcription

Abstract

The AF4/FMR2 proteins AFF1 and AFF4 act as a scaffold to assemble the Super Elongation Complex (SEC) that strongly activates transcriptional elongation of HIV-1 and cellular genes. Although they can dimerize, it is unclear whether the dimers exist and function within a SEC in vivo. Furthermore, it is unknown whether AFF1 and AFF4 function similarly in mediating SEC-dependent activation of diverse genes. Providing answers to these questions, our current study shows that AFF1 and AFF4 reside in separate SECs that display largely distinct gene target specificities. While the AFF1-SEC is more potent in supporting HIV-1 transactivation by the viral Tat protein, the AFF4-SEC is more important for HSP70 induction upon heat shock. The functional difference between AFF1 and AFF4 in Tat-transactivation has been traced to a single amino acid variation between the two proteins, which causes them to enhance the affinity of Tat for P-TEFb, a key SEC component, with different efficiency. Finally, genome-wide analysis confirms that the genes regulated by AFF1-SEC and AFF4-SEC are largely non-overlapping and perform distinct functions. Thus, the SEC represents a family of related complexes that exist to increase the regulatory diversity and gene control options during transactivation of diverse cellular and viral genes.

A Minor Subset of Super Elongation Complexes Plays a Predominant Role in Reversing HIV-1 Latency

“…In contrast, the activated elongation in JQ1-treated cells was completely gone only after the KO of AFF1 but not AFF4 (Fig. 3C), reinforcing the notion that JQ1 acts primarily by stimulating the Tat/SEC-dependent HIV-1 elongation and that the AFF1-SEC is preferentially utilized in this process (11,17,18). As for HIV-1 elongation in cells treated with either PMA or prostratin, it was similarly sensitive to the loss of AFF1 or AFF4, although the dependence of the two compounds on AFF1/4 was a few orders of magnitude lower than that of JQ1 on AFF1 (Fig.…”

“…4C). Previously, it has been shown that JQ1 activates HIV-1 transcription and reverses viral latency through mostly the Tat/SEC-dependent pathway (17,18) and that the SEC subunits ELL2 and AFF1 play an especially important role in this process (9,11). Given these revelations, it is likely that the extra ELL2 or AFF1 introduced into the AFF4 KO cells was used to assemble the Tat-friendly, ELL2/AFF1-containing SECs that efficiently promoted latency reversal in the absence of AFF4.…”

“…A typical SEC contains CDK9 and cyclin T (CycT; either CycT1 or T2), collectively referred to as P-TEFb, as well as one of each of the three pairs of homologous proteins: ELL1/ELL2, AFF1/AFF4, and ENL/AF9 (7)(8)(9). Owing to the ability of these proteins to create multiple different combinations among them, a fairly large family of related SEC complexes exists in vivo (10,11).…”

“…The ELL1/2 subunit, on the other hand, can directly increase the catalytic rate of Pol II by suppressing transient pausing (12). As these two elongation stimulatory factors act simultaneously on a single polymerase complex at the HIV-1 promoter, they synergistically boost viral transcription (9,11). In addition to P-TEFb and ELL1/2, AFF1/AFF4 is another essential SEC component due to its ability to serve as a flexible scaffold to recruit all the other subunits into a complete complex (6,13).…”

“…Compared to AFF1, AFF4 displays a greatly diminished ability to promote the Tat-P-TEFb binding because of a critical amino acid variation between the two AFF proteins (11). While this functional difference was observed mostly in HeLa and HEK293 cells, it remains to be determined whether it also exists in HIV-1's natural host, CD4 ϩ T cells.…”