Transactivation
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The family of the Nine amino acid Transactivation Domain, 9aaTAD family, comprises currently over 40 members. The 9aaTAD domains are universally recognized by the transcriptional machinery from yeast to man. We had identified the 9aaTAD domains in the p53, Msn2, Pdr1 and B42 activators by our prediction algorithm. In this study, their competence to activate transcription as small peptides was proven. Not surprisingly, we elicited immense 9aaTAD divergence in hundreds of identified orthologs and numerous examples of the 9aaTAD species' convergence. We found unforeseen similarity of the mammalian p53 with yeast Gal4 9aaTAD domains. Furthermore, we identified artificial 9aaTAD domains generated accidentally by others. From an evolutionary perspective, the observed easiness to generate 9aaTAD transactivation domains indicates the natural advantage for spontaneous generation of transcription factors from DNA binding precursors.
- MeSH
- aktivace transkripce * MeSH
- DNA vazebné proteiny chemie MeSH
- nádorový supresorový protein p53 genetika MeSH
- Saccharomyces cerevisiae - proteiny chemie MeSH
- Saccharomyces cerevisiae genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- transkripční faktory chemie MeSH
- Publikační typ
- časopisecké články MeSH
The p53 protein plays an important role in cancer prevention. In response to stress signals, p53 controls essential cell functions by regulating expression of its target genes. Full or partial loss of the p53 function in cancer cells usually results from mutations of the p53 gene. Some of them are temperature-dependent, allowing reactivation of the p53 function in certain temperature. These mutations can alter general transactivation ability of the p53 protein or they modify its transactivation only towards specific genes. We analyzed transactivation of several target genes by 23 temperature-dependent p53 mutants and stratified them into four functional groups. Seventeen p53 mutants exhibited temperature-dependency and discriminative character in human and yeast cells. Despite the differences of yeast and human cells, they allowed similar transactivation rates to the p53 mutants, thus providing evidence that functional analysis of separated alleles in yeast is valuable tool for assessment of the human p53 status.
- MeSH
- aktivace transkripce MeSH
- lidé MeSH
- mutace MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The p53 protein is a sequence-specific transcription factor controlling the expression of multiple genes and protecting cells from oncogenic transformation. In many tumors, the p53 protein is completely or partially inactivated by mutations in the p53 gene. We analyzed the transactivating activity of nine human temperature-dependent (td) p53 mutants in yeast cells. Mutations in seven of them were localized in the β-sandwich-coding region of the p53 gene, eight p53 mutants were temperature-sensitive and the R283C mutant was cold-sensitive. Patterns of their transactivation abilities towards three different responsive elements, the extent of their temperature dependency as well as discriminativity, were considerably variable. Similarly, their capacity to become reactivated by amifostine varied from complete resistance to high sensitivity. Transactivation abilities and temperature dependency of six p53 td mutants were determined in transiently-transfected H1299 human cells and revealed substantial concordance between the activity patterns of the p53 mutants in yeast and human cells. We concluded that the td p53 mutants do not comprise a uniform group, therefore, the behavior of each mutant has to be tested individually.
- MeSH
- aktivace transkripce * MeSH
- amifostin farmakologie MeSH
- geny p53 * MeSH
- kvasinky genetika MeSH
- lidé MeSH
- mutace MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- radioprotektivní látky farmakologie MeSH
- teplota MeSH
- transformace genetická MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The effect of mutations in the P53 family of transcription factors on their biological functions, including partial or complete loss of transcriptional activity, has been confirmed several times. At present, P53 family proteins showing partial loss of activity appear to be promising potential candidates for the development of novel therapeutic strategies which could restore their transcriptional activity. In this context, it is important to employ tools to precisely monitor their activity; in relation to this, non-canonical DNA secondary structures in promoters including G-quadruplexes (G4s) were shown to influence the activity of transcription factors. Here, we used a defined yeast assay to evaluate the impact of differently modeled G4 forming sequences on a panel of partial function P53 family mutant proteins. Specifically, a 22-mer G4 prone sequence (derived from the KSHV virus) and five derivatives that progressively mutate characteristic guanine stretches were placed upstream of a minimal promoter, adjacent to a P53 response element in otherwise isogenic yeast luciferase reporter strains. The transactivation ability of cancer-associated P53 (TA-P53α: A161T, R213L, N235S, V272L, R282W, R283C, R337C, R337H, and G360V) or Ectodermal Dyplasia syndromes-related P63 mutant proteins (ΔN-P63α: G134D, G134V and inR155) were tested. Our results show that the presence of G4 forming sequences can increase the transactivation ability of partial function P53 family proteins. These observations are pointing to the importance of DNA structural characteristics for accurate classification of P53 family proteins functionality in the context of the wide variety of TP53 and TP63 germline and somatic mutations.
p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.
- MeSH
- G-kvadruplexy * MeSH
- lidé MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- promotorové oblasti (genetika) genetika MeSH
- protein - isoformy genetika metabolismus MeSH
- proteiny regulující apoptózu genetika metabolismus MeSH
- protoonkogenní proteiny genetika metabolismus MeSH
- responzivní elementy genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The TP53 gene is the most frequently mutated gene in human cancer and p53 protein plays a crucial role in gene expression and cancer protection. Its role is manifested by interactions with other proteins and DNA. p53 is a transcription factor that binds to DNA response elements (REs). Due to the palindromic nature of the consensus binding site, several p53-REs have the potential to form cruciform structures. However, the influence of cruciform formation on the activity of p53-REs has not been evaluated. Therefore, we prepared sets of p53-REs with identical theoretical binding affinity in their linear state, but different probabilities to form extra helical structures, for in vitro and in vivo analyses. Then we evaluated the presence of cruciform structures when inserted into plasmid DNA and employed a yeast-based assay to measure transactivation potential of these p53-REs cloned at a chromosomal locus in isogenic strains. We show that transactivation in vivo correlated more with relative propensity of an RE to form cruciforms than to its predicted in vitro DNA binding affinity for wild type p53. Structural features of p53-REs could therefore be an important determinant of p53 transactivation function.
The p53 gene is often mutated during cancer development. Frequency and functional consequences of these mutations vary in different tumor types. We analysed conformation and temperature dependency of 23 partially inactivating temperature-dependent (td) p53 mutants derived from various human tumors in yeast. We found considerable differences in transactivation capabilities and discriminative character of various p53 mutants. No correlations in transactivation rates and conformations of the td p53 proteins were detected. Amifostine-induced p53 reactivation occurred only in 13 of 23 td mutants, and this effect was temperature dependent and responsive element specific. The most of the p53 mutations (10/13) reactivated by amifostine were located in the part of the p53 gene coding for hydrophobic beta-sandwich structure of the DNA-binding domain.
- MeSH
- aktivace transkripce genetika účinky léků genetika MeSH
- amifostin farmakologie MeSH
- financování organizované MeSH
- konformace proteinů účinky léků MeSH
- lidé MeSH
- nádorový supresorový protein p53 biosyntéza genetika chemie MeSH
- radioprotektivní látky farmakologie MeSH
- Saccharomyces cerevisiae - proteiny genetika účinky léků MeSH
- substituce aminokyselin genetika MeSH
- teplota MeSH
- Check Tag
- lidé MeSH
P53, P63, and P73 proteins belong to the P53 family of transcription factors, sharing a common gene organization that, from the P1 and P2 promoters, produces two groups of mRNAs encoding proteins with different N-terminal regions; moreover, alternative splicing events at C-terminus further contribute to the generation of multiple isoforms. P53 family proteins can influence a plethora of cellular pathways mainly through the direct binding to specific DNA sequences known as response elements (REs), and the transactivation of the corresponding target genes. However, the transcriptional activation by P53 family members can be regulated at multiple levels, including the DNA topology at responsive promoters. Here, by using a yeast-based functional assay, we evaluated the influence that a G-quadruplex (G4) prone sequence adjacent to the p53 RE derived from the apoptotic PUMA target gene can exert on the transactivation potential of full-length and N-terminal truncated P53 family α isoforms (wild-type and mutant). Our results show that the presence of a G4 prone sequence upstream or downstream of the P53 RE leads to significant changes in the relative activity of P53 family proteins, emphasizing the potential role of structural DNA features as modifiers of P53 family functions at target promoter sites.
- MeSH
- apoptóza genetika MeSH
- DNA genetika ultrastruktura MeSH
- G-kvadruplexy * MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- membránové proteiny genetika ultrastruktura MeSH
- nádorový supresorový protein p53 genetika ultrastruktura MeSH
- promotorové oblasti (genetika) genetika MeSH
- protein p73 genetika ultrastruktura MeSH
- proteiny regulující apoptózu genetika MeSH
- protoonkogenní proteiny genetika MeSH
- responzivní elementy genetika MeSH
- Saccharomyces cerevisiae genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites.
- MeSH
- aktivace transkripce genetika MeSH
- DNA chemie metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy genetika metabolismus MeSH
- konformace nukleové kyseliny * MeSH
- lidé MeSH
- mutace genetika MeSH
- mutantní proteiny metabolismus MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- oxidace-redukce MeSH
- promotorové oblasti (genetika) * MeSH
- protein HMGB1 chemie metabolismus MeSH
- proteinové domény MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
