Human cytomegalovirus IE2 both activates and represses initiation and modulates elongation in a context-dependent manner
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE193026 | GEO | 2022/05/11
REPOSITORIES: GEO
Similar Datasets
Project description:Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, in late infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that the IE2 proteins target a distinct subset of late infection HCMV promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors, which do not appreciably affect host transcription during this late time.
2020-03-05 | GSE139114 | GEO
Project description:Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provided the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs) and that DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking and to compare these results to those from standard ChIP-Seq that employs sonication and CUT&RUN which utilizes MNase to fragment the genomic DNA. The results presented indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.
2024-08-19 | GSE262859 | GEO
Project description:Transcription by RNA polymerase (Pol) II requires assembly of a preinitiation complex (PIC) composed of general transcription factors (GTFs) bound at the core promoter. In vitro, the TATA-binding protein (TBP), TFIIB, TFIIF, and Pol II are essential for PIC formation and transcriptional initiation, whereas TFIIA, TFIIE, and TFIIH are not required under certain conditions. In addition, the PIC is stable in the absence of nucleotide triphosphates, and sequential addition of GTFs results in a series of partial PICs. Here, we analyze the roles of all GTFs in yeast cells by measuring PIC formation and Pol II transcription upon depletion of individual GTFs. All GTFs are essential for TBP binding and Pol II transcription in vivo, suggesting that partial PICs composed of GTF subsets do not exist at appreciable levels. In contrast, TBP-associated factors (TAFs) contribute to Pol II transcriptional activity at most (and perhaps all) genes, but TAF-independent transcription occurs at a substantial level, particularly at promoters lacking canonical TATA elements. Lastly, unlike the case in vitro, PICs are not observed in cells deprived of uracil, and presumably UTP, suggesting a mechanism that removes transcriptionally inactive PICs from promoters.
2019-02-27 | GSE122734 | GEO
Project description:The multisubunit TFIID plays a direct role in transcription initiation by binding to core promoter elements and directing preinitiation complex assembly. Although TFIID may also function as a coactivator through direct interactions with promoter-bound activators, mechanistic aspects of this poorly defined function remain unclear. Here biochemical studies show a direct TFIID-E protein interaction that (i) is mediated through interaction of a novel E protein activation domain (AD3) with the TAF homology (TAFH) domain of TAF4, (ii) is critical for activation of a natural target gene by an E protein and (iii) mechanistically, acts by enhancing TFIID binding to the core promoter. Complementary assays establish a gene-specific role for the TAFH domain in TFIID recruitment and gene activation in vivo. These results firmly establish TAF4 as a bona fide E protein coactivator, as well as a mechanism involving facilitated TFIID binding through direct interaction with an E protein activation domain. Genome-wide profiling of mRNA levels in MEF lines with Taf4 loxp/- (ctrl), Taf4 -/- (ko), Taf4 -/- Tg:hTAF4 wt, and Taf4 -/- Tg: hTAF4 ? (TAFH-deleted).
2013-06-06 | E-GEOD-46807 | biostudies-arrayexpress
Project description:The multisubunit TFIID plays a direct role in transcription initiation by binding to core promoter elements and directing preinitiation complex assembly. Although TFIID may also function as a coactivator through direct interactions with promoter-bound activators, mechanistic aspects of this poorly defined function remain unclear. Here biochemical studies show a direct TFIID-E protein interaction that (i) is mediated through interaction of a novel E protein activation domain (AD3) with the TAF homology (TAFH) domain of TAF4, (ii) is critical for activation of a natural target gene by an E protein and (iii) mechanistically, acts by enhancing TFIID binding to the core promoter. Complementary assays establish a gene-specific role for the TAFH domain in TFIID recruitment and gene activation in vivo. These results firmly establish TAF4 as a bona fide E protein coactivator, as well as a mechanism involving facilitated TFIID binding through direct interaction with an E protein activation domain.
2013-06-06 | GSE46807 | GEO
Project description:We introduce target sites for the microRNA (miRNA) miR-142 into the 3’-untranslated region of the human cytomegalovirus (HCMV) IE2 to study the transcriptional effect of IE2 knock-down on both viral and host genes in cells that express miR-142. When comparing transcriptional data from miR-142 expressing macrophages infected with HCMV to macrophages infected with IE2-miR-142 targeted HCMV, we see a knock-down of IE2 and differential regulation of predicted viral targets of IE2 including IE1, vIL-10 and US29. We then generated fibroblasts expressing miR-142 to study the knock-down of IE2 in a different cellular system and find drastic differences in loss of IE2 on the host transcriptional profile in the two different cell types.
2017-12-28 | GSE107680 | GEO
Project description:To investigate the chromatin structure surrounding human RNA polymerase II (Pol II) transcription on host and viral genomes, chromatin immunoprecipitation (ChIP) was performed following digestion of HCMV infected primary human foreskin fibroblasts (HFFs) with DNA Fragmentation Factor (DFF). DFF is a human endonuclease responsible for cleaving between nucleosomes during apoptosis. We found that utilizing DFF as the front end for ChIP-Seq in place of sonication or Micrococcal nuclease (MNase) offered new insights into the connections between active transcription and the local chromatin on the host and that these connections were completely absent on the HCMV genome.
2022-02-17 | GSE185618 | GEO
Project description:To expand our understanding of the interplay of human RNA polymerase II (Pol II) promoter elements, promoter-proximal pausing, local chromatin structure, and co-transcriptional RNA processing, the 5′ and 3′ ends of nascent, capped transcripts and the locations of nearby nucleosomes were accurately identified. High-quality visualization tools revealed sequence elements defining the core promoter, sites of pausing, and nucleosome positioning across over 177,000 transcription start regions (TSRs). We found that cap methylation begins as transcripts attain a length of about 30 nt. A nuclear run-off assay was developed that utilizes the unique properties of the DNA fragmentation factor (DFF) to demonstrate the relative location of nucleosomes and paused Pol II. Our data support the conclusion that human Pol II promoters are functional TFIID binding sites with built-in downstream information directing ubiquitous promoter proximal pausing and the downstream nucleosome location.
2020-04-29 | GSE139237 | GEO
Project description:Rantasalo2015-Synthetic_expresion_modulator_constitutiveSTF_VP16
This model is part of a family of models describing a modular synthetic expression system that modulates the expression level of a gene in S. Cerevisae. The whole family of models is described in the article:
Synthetic transcription amplifier system for orthogonal control of gene expression in Saccharomyces cerevisiae, by
Anssi Rantasalo, Elena Czeizler, Riitta Virtanen, Juho Rousu, Harri Lähdesmäki, Merja Penttilä, Jussi Jäntti and Dominik Mojzita (submitted).
The family comprises 5 different models corresponding to the synthetic systems using:
1) the methionine induced synthetic transcription factor sTF-VP16 construct,
2) the methionine induced synthetic transcription factor sTF-B42 construct,
3) the constitutive sTF-VP16 construct,
4) the constitutive sTF-B42 construct, and
5) the constitutive sTF-VP16 construct with a different core promoter for the reporter gene.
The computational models were designed, developed and implemented by Elena Czeizler, Harri Lahdesmaki and Juho Rousu and they are all hosted separately in the Biomodels database.
The only difference between the models corresponding to the constitutive and the methionine-induced systems stands in the sTF transcription process. The only difference between the models for the systems using the sTF16 or sTF42 constructs stands in the kinetic rates associated to 2 reactions: i) the association of the polymerase with the sTF’s bound to their specific DNA sites and ii) the degradation rate of the sTF proteins corresponding to the two transcription factors sTF16 or sTF42. The first 4 models correspond to systems using the pBID2-EP core promoter for the reporter mCherry gene. Starting from the model associated to the constitutive system using the sTF16 construct we derived the 5th model corresponding to the case when the core promoter for the reporter mCherry gene is switched from pBID2-EP to pBID2-ED. The only difference between these two last models stands in the kinetic rates for the association of polymerase bound to sTF and the core promoter.
Since the 5 considered models have many parts in common, the values for the kinetic parameters corresponding to these common parts are identical in all of them.
The current model describes the gene expression modulation system using the constitutive synthetic transcription factor sTF-VP16 composed of a LexA DNA binding domain, the Herpes simplex virus transactivation domain (VP16) and a 6×His-tag. In this model, the number of boxes to which this sTF can bind (encoded in the species B) is set to 2. In the experimental setups, the number of binding boxes varied between 0 and 16 (8 binding sites on each of the 2 DNA constructs), which can be easily modified in the model by changing the initial value for B. The core promoter used for the reporter mCherry gene is pBID-EP.
All model analysis and simulations were done by using the software COPASI (Hoops, S., Sahle, S., Gauges, R., Lee, C., Pahle, J., Simus, N., Singhal, M., Xu, L., Mendes, P. and Kummer, U. (2006) COPASI- A COmplex PAthway SImulator. Bioinformatics, 22, 3067-3074.)
2016-02-23 | MODEL1510230001 | BioModels
Project description:Rantasalo2015-Synthetic_expresion_modulator_constitutiveSTF_VP16_pBID2-EDcorePromoter
This model is part of a family of models describing a modular synthetic expression system that modulates the expression level of a gene in S. Cerevisae. The whole family of models is described in the article:
Synthetic transcription amplifier system for orthogonal control of gene expression in Saccharomyces cerevisiae, by
Anssi Rantasalo, Elena Czeizler, Riitta Virtanen, Juho Rousu, Harri Lähdesmäki, Merja Penttilä, Jussi Jäntti and Dominik Mojzita (submitted).
The family comprises 5 different models corresponding to the synthetic systems using:
1) the methionine induced synthetic transcription factor sTF-VP16 construct,
2) the methionine induced synthetic transcription factor sTF-B42 construct,
3) the constitutive sTF-VP16 construct,
4) the constitutive sTF-B42 construct, and
5) the constitutive sTF-VP16 construct with a different core promoter for the reporter gene.
The computational models were designed, developed and implemented by Elena Czeizler, Harri Lahdesmaki and Juho Rousu and they are all hosted separately in the Biomodels database.
The only difference between the models corresponding to the constitutive and the methionine-induced systems stands in the sTF transcription process. The only difference between the models for the systems using the sTF16 or sTF42 constructs stands in the kinetic rates associated to 2 reactions: i) the association of the polymerase with the sTF’s bound to their specific DNA sites and ii) the degradation rate of the sTF proteins corresponding to the two transcription factors sTF16 or sTF42. The first 4 models correspond to systems using the pBID2-EP core promoter for the reporter mCherry gene. Starting from the model associated to the constitutive system using the sTF16 construct we derived the 5th model corresponding to the case when the core promoter for the reporter mCherry gene is switched from pBID2-EP to pBID2-ED. The only difference between these two last models stands in the kinetic rates for the association of polymerase bound to sTF and the core promoter.
Since the 5 considered models have many parts in common, the values for the kinetic parameters corresponding to these common parts are identical in all of them.
The current model describes the gene expression modulation system using the constitutive synthetic transcription factor sTF-VP16 composed of a LexA DNA binding domain, the Herpes simplex virus transactivation domain (VP16) and a 6×His-tag. In this model, the number of boxes to which this sTF can bind (encoded in the species B) is set to 2. In the experimental setups, the number of binding boxes varied between 0 and 16 (8 binding sites on each of the 2 DNA constructs), which can be easily modified in the model by changing the initial value for B. The core promoter used for the reporter mCherry gene is pBID2-ED.
All model analysis and simulations were done by using the software COPASI (Hoops, S., Sahle, S., Gauges, R., Lee, C., Pahle, J., Simus, N., Singhal, M., Xu, L., Mendes, P. and Kummer, U. (2006) COPASI- A COmplex PAthway SImulator. Bioinformatics, 22, 3067-3074.)
2016-02-23 | MODEL1510230003 | BioModels