The cytoplasmic domain of tomato spotted wilt virus Gn glycoprotein is required for Golgi localisation and interaction with Gc

AbstractEnvelopment of tomato spotted wilt virus nucleocapsids occurs at the Golgi stacks of infected cells. This is also the place where the two membrane glycoproteins Gn and Gc accumulate upon coexpression. The required Golgi retention signal has previously been demonstrated to reside within Gn. Using a series of truncated Gn proteins, the Golgi retention signal was mapped to a stretch of 10 amino acids on this protein's cytoplasmic tail, 20 residues downstream the transmembrane domain. Studies on the intracellular distribution of chimeric Gc proteins in which the cytoplasmic tail and/or transmembrane domain were exchanged by those from Gn, demonstrated the additional requirement of the Gn transmembrane domain for Golgi targeting. Truncated Gn constructs lacking the C-terminal 20 amino acids but still localising to the Golgi were no longer able to redirect Gc, suggesting the requirement of this domain for interaction with Gc

Role of p53 Serine 46 in p53 Target Gene Regulation

The tumor suppressor p53 plays a crucial role in cellular growth control inducing a plethora of different response pathways. The molecular mechanisms that discriminate between the distinct p53-responses have remained largely elusive. Here, we have analyzed the p53-regulated pathways induced by Actinomycin D and Etoposide treatment resulting in more growth arrested versus apoptotic cells respectively. We found that the genome-wide p53 DNA-binding patterns are almost identical upon both treatments notwithstanding transcriptional differences that we observed in global transcriptome analysis. To assess the role of post-translational modifications in target gene choice and activation we investigated the genome-wide level of phosphorylation of Serine 46 of p53 bound to DNA (p53-pS46) and of Serine 15 (p53-pS15). Interestingly, the extent of S46 phosphorylation of p53 bound to DNA is considerably higher in cells directed towards apoptosis while the degree of phosphorylation at S15 remains highly similar. Moreover, our data suggest that following different chemotherapeutical treatments, the amount of chromatin-associated p53 phosphorylated at S46 but not at pS15 is higher on certain apoptosis related target genes. Our data provide evidence that cell fate decisions are not made primarily on the level of general p53 DNA-binding and that post-translationally modified p53 can have distinct DNA-binding characteristics

The novel p53 target gene IRF2BP2 participates in cell survival during the p53 stress response

The tumor suppressor p53 contributes to the cellular fate after genotoxic insults, mainly through the regulation of target genes, thereby allowing e.g. repair mechanisms resulting in cell survival or inducing apoptosis. Unresolved so far is the issue, which exact mechanisms lead to one or the other cellular outcome. Here, we describe the interferon regulatory factor-2-binding protein-2 (IRF2BP2) as a new direct target gene of p53, influencing the p53-mediated cellular decision. We show that upregulation of IRF2BP2 after treatment with actinomycin D (Act.D) is dependent on functional p53 in different cell lines. This occurs in parallel with the down-regulation of the interacting partner of IRF2BP2, the interferon regulatory factor-2 (IRF2), which is known to positively influence cell growth. Analyzing the molecular functions of IRF2BP2, it appears to be able to impede on the p53-mediated transactivation of the p21- and the Bax-gene. We show here that overexpressed IRF2BP2 has an impact on the cellular stress response after Act.D treatment and that it diminishes the induction of apoptosis after doxorubicin treatment. Furthermore, the knockdown of IRF2BP2 leads to an upregulation of p21 and faster induction of apoptosis after doxorubicin as well as Act.D treatment

Crosstalk between c-Jun and TAp73α/β contributes to the apoptosis–survival balance

The p53-family member p73 plays a role in various cellular signaling pathways during development and growth control and it can have tumor suppressor properties. Several isoforms of p73 exist with considerable differences in their function. Whereas the functions of the N-terminal isoforms (TA and ΔNp73) and their opposing pro- and antiapoptotic roles have become evident, the functional differences of the distinct C-terminal splice forms of TAp73 have remained unclear. Here, we characterized the global genomic binding sites for TAp73α and TAp73β by chromatin immunoprecipitation sequencing as well as the transcriptional responses by performing RNA sequencing. We identified a specific p73 consensus binding motif and found a strong enrichment of AP1 motifs in close proximity to binding sites for TAp73α. These AP1 motif-containing target genes are selectively upregulated by TAp73α, while their mRNA expression is repressed upon TAp73β induction. We show that their expression is dependent on endogenous c-Jun and that recruitment of c-Jun to the respective AP1 sites was impaired upon TAp73β expression, in part due to downregulation of c-Jun. Several of these AP1-site containing TAp73α-induced genes impinge on apoptosis induction, suggesting an underlying molecular mechanism for the observed functional differences between TAp73α and TAp73β

Characterization of genome-wide p53-binding sites upon stress response

The tumor suppressor p53 is a sequence-specific transcription factor, which regulates the expression of target genes involved in different stress responses. To understand p53's essential transcriptional functions, unbiased analysis of its DNA-binding repertoire is pivotal. In a genome-wide tiling ChIP-on-chip approach, we have identified and characterized 1546 binding sites of p53 upon Actinomycin D treatment. Among those binding sites were known as well as novel p53 target sites, which included regulatory regions of potentially novel transcripts. Using this collection of genome-wide binding sites, a new high-confidence algorithm was developed, p53scan, to identify the p53 consensus-binding motif. Strikingly, this motif was present in the majority of all bound sequences with 83% of all binding sites containing the motif. In the surrounding sequences of the binding sites, several motifs for potential regulatory cobinders were identified. Finally, we show that the majority of the genome-wide p53 target sites can also be bound by overexpressed p63 and p73 in vivo, suggesting that they can possibly play an important role at p53 binding sites. This emphasizes the possible interplay of p53 and its family members in the context of target gene binding. Our study greatly expands the known, experimentally validated p53 binding site repertoire and serves as a valuable knowledgebase for future research

The novel p53 target gene IRF2BP2 participates in

cell survival during the p53 stress respons