Steven J. Triezenberg

Structure and function of transcriptional activation domains

Transcriptional activator proteins typically have distinct domains for the recognition of target genes (DNA-binding domains) and for stimulating the transcriptional machinery (activation domains). Although molecular models for the structure of activation domains are not yet available, clues are emerging from mutational analyses of many activators. Such domains may function at numerous steps in the transcription process, by making (and perhaps breaking) contacts with and among various basal transcription factors.

Genetic isolation of ADA2: A potential transcriptional adaptor required for function of certain acidic activation domains

We have devised a genetic strategy to isolate the target of acidic activation domains of transcriptional activators based on toxicity in yeast cells of the chimeric activator, GAL4-VP16. Toxicity required the integrity of both the VP16 acidic activation domain and the GAL4 DNA-binding domain, suggesting that inhibition resulted from trapping of general transcription factors at genomic sites. Mutations that break the interaction between GAL4-VP16 and general factors would alleviate toxicity and identify transcriptional adaptors, if adaptors bridged the interaction between activators and general factors. We thus identified ADA1, ADA2, and ADA3. Mutations in ADA2 reduced the activity of GAL4-VP16 and GCN4 in vivo. ada2 mutant extracts exhibited normal basal transcription, but were defective in responding to GAL4-VP16, GCN4, or the dA:dT activator. Strikingly, the mutant extract responded like wild type to GAL4-HAP4. We conclude that ADA2 potentiates the activity of one class of acidic activation domain but not a second class.

Disruption Mutations of ADA2b and GCN5 Transcriptional Adaptor Genes Dramatically Affect Arabidopsis Growth, Development, and Gene Expression

We previously identified Arabidopsis genes homologous with the yeast ADA2 and GCN5 genes that encode components of the ADA and SAGA histone acetyltransferase complexes. In this report, we explore the biological roles of the Arabidopsis ADA2b and GCN5 genes. T-DNA insertion mutations in ADA2b and GCN5 were found to have pleiotropic effects on plant growth and development, including dwarf size, aberrant root development, and short petals and stamens in flowers. Approximately 5% of the 8200 genes assayed by DNA microarray analysis showed changes of expression in the mutants, three-fourths of which were upregulated and only half of which were altered similarly in the two mutant strains. In cold acclimation experiments, C-repeat binding factors (CBFs) were induced in the mutants as in wild-type plants, but subsequent transcription of cold-regulated (COR) genes was reduced in both mutants. Remarkably, nonacclimated ada2b-1 (but not gcn5-1) mutant plants were more freezing tolerant than nonacclimated wild-type plants, suggesting that ADA2b may directly or indirectly repress a freezing tolerance mechanism that does not require the expression of CBF or COR genes. We conclude that the Arabidopsis ADA2b and GCN5 proteins have both similar and distinct functions in plant growth, development, and gene expression and may be components of both a common coactivator complex and separate complexes with distinct biological activities.

VP16-Dependent Association of Chromatin-Modifying Coactivators and Underrepresentation of Histones at Immediate-Early Gene Promoters during Herpes Simplex Virus Infection

ABSTRACT During infection by herpes simplex virus type 1 (HSV-1), the virion protein VP16 activates the transcription of viral immediate-early (IE) genes. Genetic and biochemical assays have shown that the potent transcriptional activation domain of VP16 can associate with general transcription factors and with chromatin-modifying coactivator proteins of several types. The latter interactions are particularly intriguing because previous reports indicate that HSV-1 DNA does not become nucleosomal during lytic infection. In the present work, chemical cross-linking and immunoprecipitation assays were used to probe the presence of activators, general transcription factors, and chromatin-modifying coactivators at IE gene promoters during infection of HeLa cells by wild-type HSV-1 and by RP5, a viral strain lacking the VP16 transcriptional activation domain. The presence of VP16 and Oct-1 at IE promoters did not depend on the activation domain. In contrast, association of RNA polymerase II, TATA-binding protein, histone acetyltransferases (p300 and CBP), and ATP-dependent remodeling proteins (BRG1 and hBRM) with IE gene promoters was observed in wild-type infections but was absent or reduced in cells infected by RP5. In contrast to the previous evidence for nonnucleosomal HSV-1 DNA, histone H3 was found associated with viral DNA at early times of infection. Interestingly, histone H3 was underrepresented on IE promoters in a manner dependent on the VP16 activation domain. Thus, the VP16 activation domain is responsible for recruiting general transcription factors and coactivators to IE promoters and also for dramatically reducing the association of histones with those promoters.

Curcumin inhibits herpes simplex virus immediate-early gene expression by a mechanism independent of p300/CBP histone acetyltransferase activity.

Curcumin, a phenolic compound from the curry spice turmeric, exhibits a wide range of activities in eukaryotic cells, including antiviral effects that are at present incompletely characterized. Curcumin is known to inhibit the histone acetyltransferase activity of the transcriptional coactivator proteins p300 and CBP, which are recruited to the immediate early (IE) gene promoters of herpes simplex virus type 1 (HSV-1) by the viral transactivator protein VP16. We tested the hypothesis that curcumin, by inhibiting these coactivators, would block viral infection and gene expression. In cell culture assays, curcumin significantly decreased HSV-1 infectivity and IE gene expression. Entry of viral DNA to the host cell nucleus and binding of VP16 to IE gene promoters was not affected by curcumin, but recruitment of RNA polymerase II to those promoters was significantly diminished. However, these effects were observed using lower curcumin concentrations than those required to substantially inhibit global H3 acetylation. No changes were observed in histone H3 occupancy or acetylation at viral IE gene promoters. Furthermore, p300 and CBP recruitment to IE gene promoters was not affected by the presence of curcumin. Finally, disruption of p300 expression using a short hairpin RNA did not affect viral IE gene expression. These results suggest that curcumin affects VP16-mediated recruitment of RNA polymerase II to IE gene promoters by a mechanism independent of p300/CBP histone acetyltransferase activity.

Multiple hydrophobic motifs in Arabidopsis CBF1 COOH-terminus provide functional redundancy in trans-activation.

The Arabidopsis CBF proteins activate expression of a set of genes whose upstream regulatory sequences typically harbor one or more copies of the CRT/DRE low temperature cis-acting DNA regulatory element. Using domain swap experiments in both yeast and Arabidopsis we show that the NH3-terminal 115 amino acids direct CBF1 to target genes and the COOH-terminal 98 amino acids function in trans-activation. Mutational analysis through the COOH-terminus using truncation and alanine-substitution mutants in yeast revealed four motifs that contribute positively towards activation. Overexpression of mutants in plants support this conclusion and also indicated that disruption of a single motif did not seriously compromise activity unless combined with the disruption of a second. These motifs consist of clusters of hydrophobic residues which are delimited from one another by short stretches of Asp, Glu, Pro and other residues favoring the formation of loops. This structural pattern is conserved across plant taxa as revealed through alignment of Arabidopsis CBF1 with homologous sequences from a diverse array of plant species. Overexpression in plants of the CBF1 COOH-terminus as a fusion with the yeast GAL4 DNA binding domain also resulted in severe stunting of growth, a phenotype which was alleviated if the activation domain was rendered ineffective. Taken together these results suggest that high level overexpression of an active, CBF activation domain compromises plant growth.

DA-complex assembly activity required for VP16C transcriptional activation

ABSTRACT One class of transcriptional activation domains stimulates the concerted binding of TFIIA and TFIID to promoter DNA. To test whether this DA-complex assembly activity contributes significantly to the overall mechanism of activation in vivo, we analyzed mutants of the 38-amino-acid residue VP16C activation subdomain from herpes simplex virus. An excellent correlation was observed between the in vivo activation function of these mutants and their in vitro DA-complex assembly activity. Mutants severely defective for in vivo activation also showed reduced in vitro binding to native TFIIA. No significant correlation between in vivo activation function and in vitro binding to human TATA binding protein, human TFIIB, or Drosophila melanogaster TAFII40 was observed for this set of VP16C mutants. These results argue that the ability of VP16C to increase the rate and extent of DA-complex assembly makes a significant contribution to the overall mechanism of transcriptional activation in vivo.

Two Arabidopsis Orthologs of the Transcriptional Coactivator ADA2 have Distinct Biological Functions

Histone acetylation is an example of covalent modification of chromatin structure that has the potential to regulate gene expression. Gcn5 is a prototypical histone acetyltransferase that associates with the transcriptional coactivator Ada2. In Arabidopsis, two genes encode proteins that resemble yeast ADA2 and share approximately 45% amino acid sequence identity. We previously reported that plants harboring a T-DNA insertion in the ADA2b gene display a dwarf phenotype with developmental defects in several organs. Here we describe T-DNA insertion alleles in the ADA2a gene, which result in no dramatic growth or developmental phenotype. Both ADA2a and ADA2b are expressed in a variety of plant tissues; moreover, expression of ADA2a from a constitutive promoter fails to complement the ada2b-1 mutant phenotype, consistent with the hypothesis that the two proteins have distinct biochemical roles. To further probe the cellular roles of ADA2a and ADA2b, we studied the response of the transcriptional coactivator mutants to abiotic stress. Although ada2b seedlings display hypersensitivity to salt and abscisic acid and altered responses to low temperature stress, the responses of ada2a seedlings to abiotic stress generally parallel those of wildtype plants. Intriguingly, ada2a;ada2b double mutant plants display an intermediate, gcn5-like phenotype, suggesting that ADA2a and ADA2b each work independently with GCN5 to affect genome function in Arabidopsis.

General and Specific Alterations in Programming of Global Viral Gene Expression during Infection by VP16 Activation-Deficient Mutants of Herpes Simplex Virus Type 1

ABSTRACT During productive infection by herpes simplex virus 1 (HSV-1), viral gene expression occurs in a temporally regulated cascade in which transcription of the viral immediate-early (IE) genes is strongly stimulated by the virion protein VP16. We have employed an oligonucleotide microarray to examine the effect of VP16 mutations on the overall pattern of viral gene expression following infection of HeLa cells. This microarray detects essentially all HSV-1 transcripts with relative and absolute levels correlating well with known kinetics of expression. This analysis revealed that deletion of the VP16 activation domain sharply reduced overall viral gene expression; moreover, the pattern of this reduced expression varied greatly from the pattern of a wild-type (wt) infection. However, when this mutant virus was delivered at a high multiplicity of infection or in the presence of the cellular stress inducer hexamethylene bisacetamide, expression was largely restored to the wt levels and pattern. Infection with virions that deliver wt VP16 protein at the start of infection but synthesize only truncated VP16 resulted in a normal kinetic cascade. This suggests that newly synthesized VP16 does not play a significant role in the expression of later classes of transcripts. The VP16 activation domain comprises two subregions. Deletion of the C-terminal subregion resulted in minimal changes in the level and profile of gene expression compared to a normal (wt) cascade. In contrast, deletion of the N-terminal subregion reduced the overall expression levels and skewed the relative levels of IE transcripts but did not significantly alter the kinetic pattern of early and late transcript expression. We conclude that the general activation of IE gene transcription by VP16, but not the specific ratios of IE transcripts, is necessary for the subsequent ordered expression of viral genes. Moreover, this report establishes the feasibility of microarray analysis for globally assessing viral gene expression programs as a function of the conditions of infection.

Regulation of Histone Deposition on the Herpes Simplex Virus Type 1 Genome during Lytic Infection

ABSTRACT During lytic infection by herpes simplex virus type 1 (HSV-1), histones are present at relatively low levels on the viral genome. However, the mechanisms that account for such low levels—how histone deposition on the viral genome is blocked or how histones are removed from the genome—are not yet defined. In this study, we show that histone occupancy on the viral genome gradually increased with time when transcription of the viral immediate-early (IE) genes was inhibited either by deletion of the VP16 activation domain or by chemical inhibition of RNA polymerase II (RNAP II). Inhibition of IE protein synthesis by cycloheximide did not affect histone occupancy on most IE promoters and coding regions but did cause an increase at delayed-early and late gene promoters. IE gene transcription from HSV-1 genomes associated with high levels of histones was stimulated by superinfection with HSV-2 without altering histone occupancy or covalent histone modifications at IE gene promoters. Moreover, RNAP II and histones cooccupied the viral genome in this context, indicating that RNAP II does not preferentially associate with viral genomes that are devoid of histones. These results suggest that during lytic infection, VP16, RNAP II, and IE proteins may all contribute to the low levels of histones on the viral genome, and yet the dearth of histones is neither a prerequisite for nor a necessary result of VP16-dependent transcription of nucleosomal viral genomes.