Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-κB

225

157

Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-KB. The twice-repeated KB sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Spl. We have previously shown that a cooperative interaction of NF-KB with Spl is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Spl specifically interacted with the amino-terminal region of ReIA(p65). Similarly, RelA bound directly to the zinc finger region of Spl. This interaction was specific and resulted in cooperative DNA binding to the icB and Spl sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Koxl5, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of KB-regulated genes.

Section: Methodsmentioning

confidence: 58%

An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation.

Perkins¹,

Agranoff²,

Pascal³

et al. 1994

225

157

“…Previous work on the identity of the biological targets for acidic activators in vivo has suggested that these include TBP, TFIIB, and TAF40. Although TBP has been implicated as a target for several transcriptional activators (24,25,44), the fact the TBP also directly contacts a large number of TAFs and basal transcription factors, including RNA polymerase II and TFIIB, has led to some concern as to the in vivo relevance of these in vitro interactions (20,48,50). While TAF40 has been shown to contact the VP16 activation domain (15), this contact is to the C-terminal 452-to-490 sequence and not to the more N-terminal 412-to-456 sequence that, when multimerized, is fully sufficient to activate transcription efficiently (11,41).…”

Section: Discussionmentioning

confidence: 99%

Mutational analysis of the transcription activation domain of RelA: identification of a highly synergistic minimal acidic activation module.

Blair¹,

Bogerd²,

Madore³

et al. 1994

107

124

The potent C-terminal activation domain of the RelA(p65) subunit of the cellular transcription factor NF-KB is shown to contain several discrete acidic activation modules. These short, -11-amino-acid modules were able to give rise to only a low level of transcription activation when fused to the GAL4 DNA-binding domain as monomers. However, dimers and higher-order multimers activated the transcription of minimal promoter elements as effectively as the full-length RelA or VP16 activation domain. Therefore, this 11-aminoacid ReIA-derived acidic module appears to contain all of the sequence information required to fully activate a target promoter element as long as it is presented in a form that permits functional synergy. Critical primary sequence requirements for acidic activation module function included a core phenylalanine residue and flanking bulky hydrophobic residues. Overall negative charge was necessary but not sufficient for function. While dimeric forms of the 11-amino-acid acidic activation module bound to either TFIIB or TATA-binding protein efficiently in vitro, a similarly charged peptide lacking the core phenylalanine residue failed to interact.Overall, these data demonstrate that the biological activity of the RelA activation domain is dependent on acidic activator sequences that are closely comparable to those detected in the activation domain of the viral VP16 regulatory protein. We hypothesize that the ability of these acidic activators to specifically interact with multiple components of the transcription initiation complex likely underlies the dramatic functional synergy exhibited by this class of activation domains in vivo.

“…These include the highly acidic activation domains in VP16 (50,103), p53 (10,67,72,84,97,108), c-Myc (39), v-Rel and c-Rel (55,118), and E2F-1 (37), as well as other kinds of activation domains found in the adenovirus activator ElA (48,62), the Epstein-Barr virus proteins Zta (64) and R (70), the human T-cell leukemia virus type 1 (HTLV-1) activator Taxl (8), the transactivator Tat of human immunodeficiency virus type 1 (HIV-1) (53), and human c-Fos and c-Jun (85), PU-1 (38), and Spl (20). In certain cases, reduced binding of TBP by activation domains with point mutations that reduce transactivation in vivo has provided evidence for the biological relevance of these interactions (8,50,53,62).…”

mentioning

confidence: 99%

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Xiao¹,

Pearson²,

Coulombe³

et al. 1994

339

274

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIHH and its Saccharomyces cerevisuze counterpart, factor b. The VP16-and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.Accurate transcription in vitro by human RNA polymerase II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF (RAP30 and RAP74), TFIIH (also known as BTF2), and TFIIJ (reviewed in references 13 and 121). Beginning with TFIID, which recognizes the TATA boxes present in many promoters for RNA polymerase II, these factors and RNA polymerase II can be assembled in a defined order onto a promoter (5). TFIIH and TFIIJ are the last of these factors to bind to an assembling initiation complex (12,14,26), and TFIIH, also known as BTF2, is the only factor known to possess associated enzymatic activities. These include an ATP-dependent DNA helicase activity (95, 96) and a protein kinase activity that can phosphorylate the carboxyterminal heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase 11 (12, 21,68 herpes simplex virus transactivator VP16 (107) and in the N-terminal 73 amino acids of the mammalian tumor suppressor protein p53 (23). The p53 protein has a site-specific DNA-binding domain in its carboxy-terminal portion (101). VP16 does not, by itself, bind specifically to DNA, but instead its amino-terminal portion binds DNA in association with mammalian factors, including the POU homeodomain protein Oct-1 that recognizes octamer sequences in DNA (28,60,102). When fused to the DNA-binding domain of GAL4, the VP16 and p53 activation domains stimulate transcription in yeast and human cells of a gene bearing GAL4-binding sites (9,16,23,74,87,92). These observations imply that common mechanisms for transcriptional activation by acidic activators may exist in fungi and mammals.Many activation domains have been shown to interact with the TATA-box-binding protein (TBP) subunit of TFIID. These include the highly acidic activation domains in VP16 (50,103), p53 (10,67,72,84,97,108),118), and E2F-1 (37), as well as other kinds of activation domains found in the adenovirus activator ElA (48, 62), the Epstein-Barr virus proteins Zta (64) and R (70), the human T-cell leukemia virus type 1 (HTLV-1) activator Taxl (8), the transactivator Tat of human immunodeficiency virus type 1 (HIV-1) (53), and human c-Fos and c-Jun (85), PU-1 (38), and Spl (20). In certain cases, reduced binding of TBP by activation domains wit...