TAF10 and TAF10b partially redundant roles during Drosophila melanogaster morphogenesis.

Transcription of eukaryotic genes requires the cooperative action of the RNA polymerase complex, the general transcription factors (TFIIB, TFIID, TFIIE, TFIIF and TFIIH) and chromatin modifiers. The TFIID complex contributes to transcriptional activation by several mechanisms and has a subunit with associated histone acetyltransferase (HAT) activity. The histone modifier SAGA complex has both HAT and deubiquitylase (DUB) activities. TFIID and SAGA share several TBP-associated factors (TAFs), but not their HAT subunit. Recently, several duplicated TAF proteins have been identified in higher eukaryotes, but their functional diversity has been so far poorly characterized. Here, we report the functional similarities and differences of TAF10 and TAF10b, the two TAF10 orthologs of Drosophila melanogaster. Results from in silico modeling suggest that dTAF10 and dTAF10b have similar secondary structures characterized by the presence of a histone-fold domain. Additionally, dTAF10 and dTAF10b share interaction partners and show similar expression patterns in neuronal tissues. Nonetheless, dTAF10 and dTAF10b seem to have partly distinct functions. To investigate their roles, we generated dTaf10-dTaf10b double-mutants and rescued the mutant flies with transgenes, which allowed the translation of either dTAF10 or dTAF10b protein. We found that the loss of dTAF10b resulted in pupal lethality, while animals lacking dTAF10 were able to form puparium. dTaf10 mutant adults showed distorted eye morphology. During DNA repair, dTAF10 and dTAF10b act redundantly, suggesting that these proteins have distinct but partially overlapping functions.

In vivo effects of abolishing the single canonical sumoylation site in the C-terminal region of Drosophila p53.

Using yeast two-hybrid screens we determined that Drosophila (Dm)p53 interacts with proteins involved in sumoylation (UBA2, UBC9 and PIAS) through different regions of its C-terminal domain. A K302R point mutation within a single canonical sumoylation site of Dmp53 did not abolish the observed interactions. These observations prompted us to analyze whether Dmp53 sumoylation at this site has any functional role in vivo. Genetic assays showed that deleting one copy of genes involved in sumoylation (lwr, Su(var)2-10 or smt3 heterozygosity) enhanced slightly the mutator phenotype of Dmp53. We compared the in vivo effects of wild type and K302R Dmp53 overproduced from transgenes and determined that similar levels of expression of the mutant and wild type proteins resulted in similar phenotype, and the two proteins showed similar cellular localization. The half life and the trans-activator activity of K302R mutant and wild type Dmp53 were also comparable. Lastly, by analyzing wild type and K302R Dmp53 expressed at different levels in animals and in S2 cells we detected no differences between the mobility of the mutant and wild-type protein. From these data we conclude that under normal developmental conditions the loss of SUMO modification at K302 does not affect Dmp53 function significantly.

Misregulated RNA Pol II C-terminal domain phosphorylation results in apoptosis.

Misregulation of the level of RNA polymerase II carboxyl-terminal domain (CTD) phosphatase, Fcp1, in Drosophila results in high level of caspase-mediated apoptosis. Apoptosis induction by Fcp1 misregulation requires the presence of Drosophila melanogaster (Dm)p53, but occurs without the transcriptional activation of Dmp53 proapoptotic targets rpr, ark, and hid. Overproduction of a transcription activation-defective mutant Dmp53 protein increases, while Dmp53 null background decreases significantly the level of apoptosis in Fcp1-misregulated animals. Generating the apoptotic signal does not require the function of the ATM and Rad3-related kinase (ATR), and no significant level of nucleo-cytoplasmic translocation of Dmp53 is detectable in cells expressing Fcp1 at an abnormal level. Immunostaining of larval salivary gland polytene chromosomes with anti-Dmp53 antibodies indicates Dmp53 localization at several transcriptionally active chromosomal regions in wild-type cells, while in Fcp-misregulated cells the association of Dmp53 with specific chromosomal sites is decreased.

Different sets of genes are activated by p53 upon UV or ionizing radiation in Drosophila melanogaster.

The p53 tumour suppressor plays central role in the maintenance of genome integrity. P53 deficient fruit flies are highly sensitive to ionizing radiation (IR) and show genome instability suggesting that the Drosophila melanogaster p53 (Dmp53) is necessary for the proper damage response upon IR. We found that Dmp53 null fruit flies are highly sensitive to ultraviolet radiation (UV) as well. We analyzed the expression levels of apoptotic genes in wild type and Dmp53 null mutant animals after UV or IR using quantitative real-time RT-PCR. Ark (Apaf-1 related killer) was induced in a Dmp53-dependent way upon UV treatment but not by IR, hid (head involution defective/wrinkled) was induced upon both types of DNA damage, while reaper was induced only upon IR but not UV treatment. Using microarray analysis we identified several further genes that are activated upon UV irradiation in the presence of wild type Dmp53 only. Some but not all of these genes show Dmp53-dependent activation upon IR treatment as well. These results suggest that Dmp53 activates distinct cellular pathways through regulation of different target genes after different types of DNA damage.

Interaction of bovine leukemia virus transactivator Tax with bZip proteins.

The bovine leukemia virus transactivator, BLV Tax, augments transcription from three imperfect 21-bp repeats in the viral transcriptional regulatory region. Each BLV 21-bp repeat contains a cAMP response element (CRE) in the center and unique 5' and 3' neighboring sequences which are crucial for the transcriptional activation by BLV Tax. Here we describe the interactions of recombinant BLV Tax with cellular bZip proteins. The recombinant BLV Tax, tagged at the carboxy terminus with a hexahistidine extension, was prepared by solubilization in 6 M guanidine hydrochloride and renaturation on the Ni(2+)-chelating Sepharose gel matrix. The renatured BLV TaxH6 activates the BLV LTR when introduced into HeLa cells by scrape loading. Furthermore, the purified BLV TaxH6 enhances binding of members of the CREB/ATF family of bZip proteins to CRE motifs by interacting with their bZip domains in vitro. Chemical cross-linking indicates that dimerization of bZip proteins such as CREB becomes greatly facilitated in the presence of BLV Tax. These results suggest that BLV Tax interacts directly with CREB/ATF-like factors to activate viral mRNA transcription.

In vitro selection of DNA elements highly responsive to the human T-cell lymphotropic virus type I transcriptional activator, Tax.

The human T-cell lymphotropic virus type I (HTLV-I) transactivator, Tax, the ubiquitous transcriptional factor cyclic AMP (cAMP) response element-binding protein (CREB protein), and the 21-bp repeats in the HTLV-I transcriptional enhancer form a ternary nucleoprotein complex (L. J. Zhao and C. Z. Giam, Proc. Natl. Acad. Sci. USA 89:7070-7074, 1992). Using an antibody directed against the COOH-terminal region of Tax along with purified Tax and CREB proteins, we selected DNA elements bound specifically by the Tax-CREB complex in vitro. Two distinct but related groups of sequences containing the cAMP response element (CRE) flanked by long runs of G and C residues in the 5' and 3' regions, respectively, were preferentially recognized by Tax-CREB. In contrast, CREB alone binds only to CRE motifs (GNTGACG[T/C]) without neighboring G- or C-rich sequences. The Tax-CREB-selected sequences bear a striking resemblance to the 5' or 3' two-thirds of the HTLV-I 21-bp repeats and are highly inducible by Tax. Gel electrophoretic mobility shift assays, DNA transfection, and DNase I footprinting analyses indicated that the G- and C-rich sequences flanking the CRE motif are crucial for Tax-CREB-DNA ternary complex assembly and Tax transactivation but are not in direct contact with the Tax-CREB complex. These data show that Tax recruits CREB to form a multiprotein complex that specifically recognizes the viral 21-bp repeats. The expanded DNA binding specificity of Tax-CREB and the obligatory role the ternary Tax-CREB-DNA complex plays in transactivation reveal a novel mechanism for regulating the transcriptional activity of leucine zipper proteins like CREB.