TRANSCRIPTIONAL ACTIVATION

Top PDF TRANSCRIPTIONAL ACTIVATION:

Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein.

Identification of two independent transcriptional activation domains in the Autographa californica multicapsid nuclear polyhedrosis virus IE1 protein.

(Fig. 4B). In construct CDE, the removal of region C resulted in a small but detectable increase in the level of activation by DE (Fig. 3B). It is possible that the basic charges within re- gions C and E may negatively affect transcriptional activation. Transcriptional inhibition domains that are constituted from positively charged amino acids are known to exist in transcrip- tional activators such as the Bel1 transcriptional activator of human foamy virus (8, 34). It has been reported that IE1 negatively regulates transcription from the ie0, ie2, and pe38 promoters (7, 30, 35). Therefore, regions C (G123-A168) and E (G222-T266) may play a role in negative regulation by IE1. Mechanisms that may explain the observed inhibition by these basic regions could include (i) direct interactions with compo- nents of the assembled RNA polymerase II complex, (ii) indi- rect interactions through secondary “inhibitor” proteins, or (iii) neutralization of adjacent activation regions on the IE1 protein.
Show more

9 Read more

Transcriptional Activation Domains of the Candida albicans Gcn4p and Gal4p Homologs

Transcriptional Activation Domains of the Candida albicans Gcn4p and Gal4p Homologs

Definition of the C. albicans Gcn4p (CaGcn4p) activation domain. We have recently performed a detailed genome an- notation of the C. albicans genome (6), which showed that frequently the transcription factors of this organism share ho- mology to transcription factors of other organisms only within the DNA binding domain. We have defined 198 S. cerevisiae genes whose products contain a DNA binding domain and are classified as transcription factors by combining the list of tran- scriptional regulators of Harbison et al. (23) with the list from http://www.yeastract.com/tflist.php. Of these, 32 were experi- mentally shown to be transcriptional repressors in S. cerevisiae. Ninety-nine of the remaining 166 S. cerevisiae transcriptional activators were found to have C. albicans homologs, half of which share homology only within a DNA binding domain (Fig. 1). A detailed assessment of global and transcriptional activation domain similarities is provided (see Table S1 in the supplemental material). Since there is no primary sequence that defines the activation domain as a module, the nature of the activation domain is based on the experimentally defined part of the transcription factor. A majority of S. cerevisiae transcription factors, such as Gal4p, Gcn4p, Upc2p, Leu3p, and Arg81p (11, 13, 26, 28, 38, 46, 58), were experimentally shown to have acidic activation domains. When we compared the transcription factors of S. cerevisiae with the transcription factors of C. albicans, we observed that in some cases the sequence of the experimentally defined activation domains of
Show more

11 Read more

Molecular mechanism of transcriptional activation by oestrogen receptor alpha

Molecular mechanism of transcriptional activation by oestrogen receptor alpha

Repeated attempts to isolate D rosophila p i 60 hom olog by expression screening or by amplification of cDNA with degenerate oligonucleotides were unsuccessful. Indeed, sequence homology searches were unable to identify p i 60- like proteins in the Drosophila genome (Adams et al., 2000). However, the notion that p i 60 coactivators may be unique to vertebrates has recently been challenged. A novel Drosophila gene, named taiman (tai), was identified in a genetic screen for m utations that cause m igration defects to border cells (Bai et al., 2000). The Drosophila ovary consists of egg chambers in which border cells are found. During oogenesis, the border cells migrate from the anterior tip of the egg chamber to the border of the oocyte through the interior of the chamber (Spradling, 1993). Loss of function mutation in the tai gene causes the border cells to migrate at a slower rate. Surprisingly, molecular cloning of the tai gene revealed that it may be a functional homologue to the mammalian p i 60 coactivators (Bai et al., 2000). The predicted TAI protein contains a PAS domain at its N-terminus, three NR interacting motifs (two LXXLL and one LXXML motif) in the central region and a glutamine rich region at the C-terminus which may serve as transcriptional activation domain. The PAS domain is the only region which can be aligned with confidence owing to extremely low sequence homology between the human p i 60 proteins with TAI (Appendix I, Figure AI.4). Even for the PAS domain, the human p i 60 coactivators appear to share a higher degree of similarity with Drosophila ARNT, SIM and aryl hydrocarbon receptor than TAI. It is therefore not surprising that TAI has eluded from sequence homology searches.
Show more

253 Read more

Regulation of Hepatitis C Virus Replication by Nuclear Translocation of Nonstructural 5A Protein and Transcriptional Activation of Host Genes

Regulation of Hepatitis C Virus Replication by Nuclear Translocation of Nonstructural 5A Protein and Transcriptional Activation of Host Genes

mutation in the NS5B RNA-dependent RNA polymerase. SGR-NS5A-vX are subgenomic replicons carrying NS5A variant sequence vX; SGR-Nim is the wt replicon (24); SGR-GND is the nonreplicative control replicon. NTR, nontranslated region; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; HDV, hepatitis delta virus. (B) NS5A protein expression in NG cells transfected with subgenomic replicons encoding different NS5A variants as analyzed by Western blotting at 96 h posttransfection. Actin expression was used as a loading control. Bands corresponding to 56-kDa NS5A or 42-kDa actin are indicated. (C) NS5A protein expression in NG cells transfected with subgenomic replicons encoding NS5A-v3, -v4, and -v5 was analyzed by Western blotting at 4 and 96 h posttransfection. Actin expression was used as a loading control. Bands corresponding to 56-kDa NS5A or 42-kDa actin are indicated. (D) NG cells transduced with NS5A-v1 and -v5 lentiviral vectors were treated with cycloheximide (CHX) and analyzed for NS5A expression by Western blotting at 0 and 96 h posttreatment. (E) Replication efficiency of HCV subgenomic replicons carrying different NS5A variants was analyzed in the cell lysates as detailed in panel B. Luciferase activities were assayed in transfected cells, and the replication efficiency was calculated as a percentage of that obtained with replicon SGR-Nim (represented by the dashed line). The data are indicative of mean ⫾ SEM replication efficiencies obtained from 7 independent experiments carried out in triplicate. (F) Replication efficiency of HCV subgenomic replicons carrying the v1 and v5 NS5A variants was analyzed as a time course in the cell lysates. Luciferase activities were assayed in transfected cells and are presented as a percentage of that obtained at 4 h. Data are indicative of mean ⫾ SEM replication efficiencies obtained from 4 independent experiments. (G) Relationship between the transcriptional activation capacities of NS5A variants and the replication efficiencies of the corresponding subgenomic replicons. Data points represent the mean transactivational capacities and replication efficiencies for each variant.
Show more

18 Read more

Transcriptional activation by the mouse oestrogen receptor

Transcriptional activation by the mouse oestrogen receptor

To assess if the amounts of functional wild-type and mutant receptor proteins in transfected cells were similar the expression plasmids were transfected into COS-1 cells and extracts from these cells tested for the presence of receptor in a band shift assay. For transfection cells were collected and divided into equal aliquots, using this method the transfection efficiencies, monitored by luciferase activity derived from the internal control plasmid, in any batch were very similar. The protein concentration of each extract was determined and these data used to normalise the amount of extract analysed in the band-shift assay. The results shown in Figure 3.6 indicated that all receptor proteins retained high affinity DNA binding and that alterations in receptor levels were not sufficient to account for the changes in transcriptional activation. The transfections were repeated three times with similar results. The kinetics of DNA binding by the mutant receptors has not been examined but all the mutant receptors retain the part of the protein shown by Fawell et a i, (1990a) to be sufficient to bind DNA with nearly wild-type affinity. Similar results were obtained using mutant receptor proteins synthesised in vitro (data not shown). These data suggest that the mutant receptor proteins retain the ability to bind DNA with high affinity.
Show more

216 Read more

dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

of these transcription factor binding motifs are amenable to dCas-mediated transcriptional activation and whether there are interactions between the endogenous transcription factors and the synthetic transactivator is unclear. A systematic tar- geting of gRNA along the TIMP promoters demonstrated that some sites were hot-spots leading to significant induction of promoter activity while others were not. Furthermore, gRNA targeting the top- or the bottom-strand of the same promoter region made a large difference in the level of induction. Overall, no apparent rule emerged that allowed identifica- tion of the best gRNA target sites for induction of the TIMP promoters with regard to proximity to known transcription factor binding motifs, affirming the necessity of adopting a trial and error approach in identifying the optimal gRNA target site. Nevertheless, a systematic survey of the gRNA targets along the TIMP promoters identified several candi- dates capable of TIMP induction by the promoter-reporter assay and recapitulated in a cellular context.
Show more

11 Read more

Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcrip- tional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to ,1% of the wild- type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.
Show more

9 Read more

Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene In curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells

Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene In curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells

Previous study revealed that curcumin activated AMPK and subsequently inhibited the activation of NF-κB in human colon cancer cells, demonstrating that curcumin suppressed NF- κB via AMPK activation [42]. Pan et al also reported that curcumin induces AMPK activation in ovarian cancer cells [43]. The present study clearly indicated that curcumin triggered transcriptional activation of hST8Sia I gene via AMPK signaling pathway in A549 cells, as demonstrated by AMPK inhibitor. Our present finding is consistent with the previous study showing curcumin-induced autophagy via activating AMPK signaling pathway in A549 cells [19]. In contrast with previous finding demonstrating suppression of NF-κB via AMPK activation by curcumin in human colon cancer cells [42], however, our data indicate that in curcumin-stimulated A549 cells transcriptional activation of hST8Sia I gene induces by activation of NF-κB via AMPK signaling pathway. These results suggest that suppression or activation of NF-κB via AMPK signaling pathway by curcumin can be varied depending upon the types of cells.
Show more

23 Read more

In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila

In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila

ABSTRACT A number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA ef fi cacy for any given gene, and we con fi rm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.
Show more

23 Read more

Transcriptional activation and dimerisation properties of oestrogen receptor β

Transcriptional activation and dimerisation properties of oestrogen receptor β

The oestrogen receptor (ER) is a m em ber of the nuclear receptor superfam ily of ligand inducible transcription factors. In the absence of oestrogen the ER exists in the cell as p a rt of an inactive complex w ith heat shock proteins. U pon binding oestrogen the heat shock proteins are displaced, a if the ER is able to bind DNA as a dim er w here it stim ulates transcription from oestrogen responsive elem ents (ERE) in the vicinity of target genes. The ER exists in two forms, the classical ERa and the recently discovered ERp, which are encoded by distinct genes. ERP has a sim ilar b in d in g affinity for 17p-oestradiol as ERa and is capable of activ atin g tran scrip tio n from ERE containing prom oters. W hen co­ expressed in v itr o or in v iv o ERa and ERP form heterodim ers, w hich bind to an ERE w ith an affinity similar to that of ERa hom odim ers but g reater than that of ERp h o m o d im e r s . The h etero d im er, like the hom odim ers, are capable of binding the steroid receptor coactivator 1 (SRCl) w hen b ound to DNA and stim ulating transcription from ERE containing reporter genes in cell lines. ERa has two well characterised transcriptional activation dom ains, activation function 1 (API) in the N -term in u s and activation function 2 (AF2) in the C -term inus. A com parison of the A Fl and AF2 dom ains from ERa and ERp revealed that ERp does not have an A Fl activity equivalent to that of ERa, while their AF2 activities are similar.
Show more

193 Read more

Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

Retroviral insertional activation in a herpesvirus: transcriptional activation of US genes by an integrated long terminal repeat in a Marek's disease virus clone.

MDV, where the vast majority of the stable LTR insertions are localized (15). This finding has now been corroborated by at least two other studies involving coinfection of HVT or MDV with avian leukosis virus (8a, 13). Such a clustered integration pattern contrasts with the generally nonspecific nature of ret- roviral insertions but is reminiscent of the region-specific inte- grations near proto-oncogenes in leukemias and lymphomas (for a review, see reference 19). In the latter case, tumors arise presumably as a result of selection for insertions that success- fully activate proto-oncogenes. We do not know what the se- lective pressures for the LTR insertion clusters in MDV are but have considered several possibilities (15). One possibility is that REV LTR-mediated insertional activation of MDV genes near the R S /U S junctions provides a selective growth advan-
Show more

8 Read more

Transcriptional activation of USP16 gene expression by NFκB signaling

Transcriptional activation of USP16 gene expression by NFκB signaling

codon, and translation frame. USP16 has been reported to contribute to the somatic stem-cell defects in DS and reduce the self-renewal of multiple somatic stem cells [17], suggesting that some of the pathological features associated with DS may result from a stem-cell imbal- ance due to overexpression of USP16 . It was first identi- fied as a histone H2A specific deubiquitinase that regulates cell cycle progression and gene expression in human cells [18]. This deubiquitinating enzyme, USP16 , removes the ubiquitin protein from H2A-K119, and upregulates the transcription of the Ink4a locus [17]. The Ink4a locus encodes the p16 Ink4a and the p19 Arf genes, which are important members participating in self-renewal and senescence pathways. It was reported that USP16 was upregulated in response to DNA dam- age, and the upregulation of its expression was HECT and RCC1-like domain-containing protein 2 (HERC2)- dependent [19]. Furthermore, USP16 was shown to regulate embryonic stem cell gene expression and hematopoietic stem cell function [20, 21]. A recent study reported that USP16 was involved in cancer, and its downregulation promoted hepatocellular carcinoma cells growth [22]. The converging lines of evidence shed light on USP16 ‘s functions, but the transcriptional regulation of USP16 gene is largely unknown.
Show more

12 Read more

Genetic evidence for transcriptional activation by the yeast IME1 gene product.

Genetic evidence for transcriptional activation by the yeast IME1 gene product.

Subcellular distribution of an fusion protein: T h e idea that IMEl is a transcriptional acti- vator predicts that IMEl should be a nuclear protein. We used indirect immunofluorescence to examine the subcellular distribution of an fusion pro- tein containing IMEl residues 1-340. T h e fusion permitted sporulation of an diploid, indi- cating that it had IMEl function. T h e fusion protein was concentrated in the nucleus (Figure 7). A control fusion to the first t w o codons was distrib- uted throughout the cells (data not shown). These results suggest that IMEl is a nuclear protein.
Show more

10 Read more

Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter

Molecular mechanism of transcriptional activation of angiotensinogen gene by proximal promoter

acetyltransferase (CAT) assays with 5'-deleted constructs showed that the proximal promoter region from -96 to +22 of the transcriptional start site was enough to express HepG2-specific CAT activity. Electrophoretic mobility shift assay and DNase I footprinting demonstrated that the liver- and HepG2-specific nuclear factor (angiotensinogen gene- activating factor [AGF2]) and ubiquitous nuclear factor (AGF3) bound to the proximal

11 Read more

Mechanisms of Inhibition of Nuclear Hormone Receptor-Dependent Hepatitis B Virus Replication by Hepatocyte Nuclear Factor 3β

Mechanisms of Inhibition of Nuclear Hormone Receptor-Dependent Hepatitis B Virus Replication by Hepatocyte Nuclear Factor 3β

HBV RNA synthesis and viral replication has been investi- gated in mouse fibroblasts. HNF3␤ is a member of the hepa- tocyte nuclear factor 3/forkhead transcription factor family (19, 21, 23). The members of this family of transcription factors are characterized by a conserved winged helix DNA binding domain that is approximately 100 amino acids in length (8, 19, 21). The HNF3 polypeptides have additional conserved se- quences in the amino- and carboxyl-terminal regions, flanking the DNA binding domain that is located in the middle of the polypeptide (19). In the case of HNF3␤, these conserved amino acid sequences have been shown to comprise part of the transcriptional activation domains of this polypeptide (32, 33). In this analysis, the amino-terminal transcriptional activation domain of HNF3␤ (32, 33) was shown to be primarily respon- sible for inhibiting viral replication, whereas the carboxyl-ter- minal transcriptional activation domain of HNF3␤ (32, 33) did not greatly affect HBV DNA synthesis. The inhibitory effect of this HNF3␤ domain on viral replication contrasts with the observation that the amino-terminal transcriptional activation domain was responsible for the increase in reporter gene ex- pression mediated by the HBV large surface antigen and nu- cleocapsid promoters. These results suggested that the lower level of the 3.5-kb HBV RNA might be due to HNF3␤ reduc- ing the rate of 3.5-kb HBV RNA elongation rather than neg- atively regulating nucleocapsid promoter activity. This possi- bility was supported by the observation that HNF3␤ could reduce viral replication when pregenomic RNA was synthe- sized from the cytomegalovirus (CMV) immediate-early pro- moter rather than the HBV nucleocapsid promoter. In addi- tion, the ability of HNF3␤ to preferentially decrease the level of the pregenomic RNA compared with precore RNA pro- duced conditions where hepatitis B e antigen (HBeAg) also contributed to the reduction in viral biosynthesis. Therefore, it appears that HNF3␤ inhibits HBV replication by reducing * Corresponding author. Mailing address: Department of Cell Biol-
Show more

10 Read more

Brd4-Independent Transcriptional Repression Function of the Papillomavirus E2 Proteins

Brd4-Independent Transcriptional Repression Function of the Papillomavirus E2 Proteins

The low-risk HPV11 E2 transcriptional repression function is also independent of Brd4. In contrast to our results pre- sented here, a recent study has implicated a role for Brd4 in the transcriptional silencing function of HPV11 E2 (73). Sim- ilar to HPV16 E2 and HPV18 E2, the low-risk HPV11 E2 protein also has transactivation properties and can repress the E6/E7 promoter in the HPV11 LCR (11, 28). We therefore examined the possibility that Brd4 might specifically be in- volved in the E2 repression functions of HPV11. Using a lu- ciferase reporter assay with the HPV11 LCR positioned up- stream of the luciferase gene, we found that HPV11 E2 repressed the basal activity of the reporter construct by ap- proximately 60% in the presence of control shRNA-scr (Fig. 7B, lanes 1 and 2) and that the Brd4 knockdown had no significant effect on the repression capacity of HPV11 E2 (Fig. 7B, lanes 3 and 4). Simultaneously performed Brd4 shRNA knockdown experiments resulted in a strong inhibition of BPV1 E2 transactivation functions, indicating that the knock- down was sufficient to inhibit E2 functions (Fig. 7A). In addi- tion, we examined the effect of the Brd4 CTD on the HPV11 E2 transcriptional repression function (Fig. 7C). We observed that HPV11 E2 repressed the HPV11 LCR promoter (Fig. 7C, lanes 1 and 2) and that the coexpression of the Brd4 CTD did not rescue the repression of the E6/E7 promoter (Fig. 7C, lanes 3 and 4). This result is consistent with observations for the HPV18 LCR. The slight increase of E2-dependent tran- scriptional repression in the presence of the Brd4 CTD is likely due to the effect of the Brd4 CTD on stabilization of the E2 protein (34). Thus, even though Brd4 is required for the phys- iologic transcriptional activation function of BPV1 E2 (Fig. 7A), it is not required for the transcriptional repression func- FIG. 5. Repression of HPV18 transcription and stabilization of p53
Show more

11 Read more

How the Rgt1 Transcription Factor of Saccharomyces cerevisiae Is Regulated by Glucose

How the Rgt1 Transcription Factor of Saccharomyces cerevisiae Is Regulated by Glucose

2. Residues 320–390 contribute to transcriptional acti- However, Std1 and Mth1 have only partially overlapping vation by Rgt1. None of these deletion mutations roles in regulating Rgt1 function: Mth1, but not Std1, abolish transcriptional activation, suggesting that is required for DNA binding by Rgt1 and inhibits phos- multiple regions of Rgt1 contribute to this function. phorylation of Rgt1 (Kim et al. 2003). Std1 seems to We were unable to produce an Rgt1 missing all of negatively regulate Rgt1-mediated transcriptional acti- its transcriptional activation domains, because most vation [like Gal80 masks Gal4 activation (Lue et al. 1987; of the many large deletion mutations of RGT1 that we Ma and Ptashne 1987a; Chasman and Kornberg constructed were undetectable in yeast cell extracts 1990)].
Show more

12 Read more

Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives

Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives

Due to the cellular complexity of eukaryotic cells, the choice of reporter gene determines the background noise and the approach of how to characterize the biosensors. Because of the simplicity, high sensitivity and low back- ground noise, ATP-independent luciferase (NanoLuc) is a promising reporter to characterize eukaryotic tran- scriptional systems. Mutagenesis, directed evolution and computational tools are important to engineer novel DNA-binding and effector-binding domains. Promoter strength, the position and number of activator or repres- sor-biding sites are critical to tune the dynamic response, the sensitivity and specificity of engineered sensors. Repression-based transcriptional regulation is rare in eukaryotic cells, a transcriptional activation domain is generally required to translate a bacteria repressor to a MRTF sensor in eukaryotic cells. To enhance the sen- sitivity and the detection limit, one should always con- sider using a minimal promoter containing only the core TF binding sites and the TATA box. Nuclear import and export of transcriptionally active MRTFs plays a criti- cal role in regulating the sensor activity. The molecular underpinnings and structure of nuclear localization sig- nal (NLS), nuclear export sequence (NES) and tran- scriptional activation domain were exemplified with a yeast-derived ROS sensor Yap1 and Skn7. Rational design of novel MRTF sensors is possible via computational approach and protein evolution.
Show more

13 Read more

Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the inter- actions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.
Show more

13 Read more

The EBNA1 Protein of Epstein-Barr Virus Functionally Interacts with Brd4

The EBNA1 Protein of Epstein-Barr Virus Functionally Interacts with Brd4

tional activation without affecting other EBNA1 functions (45, 58). Therefore, we were particularly interested in assessing the roles of these sequences in the Brd4-mediated segregation assay. We found that the 61–83 deletion abrogated Brd4-me- diated segregation while the 325–376 deletion had only a small effect, indicating that the interaction with Brd4 occurred pre- dominantly through transcriptional activation sequences. This result was also supported by coimmunoprecipitation experi- ments in human cells, where EBNA1 ⌬ 325–376 bound EBNA1 to the same degree as did wild-type EBNA1, but no binding was detected with EBNA1 ⌬ 61–83. One major difference in these two assays was seen with EBNA1 ⌬ 8–67, which is partially defective in both transcriptional activation and segregation in human cells (58). This mutant did not support Brd4-mediated segregation in yeast but was found to bind Brd4 better than wild-type EBNA1 in human cells. Since the 8–67 deletion did not increase the expression level of EBNA1, the increased binding to Brd4 indicates that removal of residues 8 to 67 allows greater access or more stable binding of Brd4 to EBNA1 sequence 61–83. However, this did not translate into more efficient segregation in the yeast system, and we suggest two possible reasons for this discrepancy. First, Brd4 binding may not be the only requirement for segregation in yeast and sequence 8–67 might provide an important role in segregation that is independent of Brd4 binding. Second, constitutive or unusually strong binding of EBNA1 to Brd4 may be detrimen- tal to plasmid maintenance, especially if the Brd4-EBNA1 interaction usually occurs transiently. For example, since Brd4 is known to associate with chromatin throughout the cell cycle,
Show more

11 Read more

Show all 10000 documents...