Human cytomegalovirus IE2 both activates and represses initiation and modulates elongation in a context-dependent manner
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ABSTRACT:
PROVIDER: PRJNA794151 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17429659_1.fastq.gz | Fastqsanger.gz | |||
| SRR17429659_2.fastq.gz | Fastqsanger.gz | |||
| SRR17429660_1.fastq.gz | Fastqsanger.gz | |||
| SRR17429660_2.fastq.gz | Fastqsanger.gz | |||
| SRR17429661_1.fastq.gz | Fastqsanger.gz |
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Project description:Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-ChIP approach that informs on local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms involving direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within the target core promoter region, blocking the assembly preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 activates transcription by binding proximal to, but not within, core promoter regions. In addition, evidence is provided that IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 is demonstrated to be important for the synthesis of a spliced RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 only sparingly engages the host genome.
2022-05-11 | GSE193026 | GEO
Project description:Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.
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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.
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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:Human cytomegalovirus induces neuronal gene expression to promote virion maturation in macroH2A1-dependent manner
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Project description:miRNA-mediated targeting of human cytomegalovirus reveals biological host and viral targets of IE2
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Project description:Decoding murine cytomegalovirus
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Project description:Cell type differences in the human cytomegalovirus (HCMV) transcriptome may result from variations in transcription and/or post-transcription. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DFF ChIP-Seq, rapid viral protein degradation, and promoter mutation and function assays). In D-NT2, but not fibroblasts, RNA polymerase II initiation and elongation at several viral promoters requires viral DNA synthesis and are independent of host P-TEFb, viral IE2, or viral UL87 late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase transcription and mutations in CREB and NF-kB response elements decrease transcription. The GC-box sequence mutations also alter infected cell morphology and gene expression program, whereas mutations in CREB and NF-kB response elements do not. In D-NT2, LTF-driven promoters constitute a smaller proportion of the viral late promoter population and are generally less active. Additionally, viral genomes have more nucleosomes, potentially restricting LTF access. A TBP-IE2-nucleosome complex, with more nucleosome than in fibroblasts, covers the MIE promoter transcription start site, potentially contributing to epigenetically silence of the promoter.
2025-07-22 | GSE291633 | GEO
Project description:Cell type differences in the human cytomegalovirus (HCMV) transcriptome may result from variations in transcription and/or post-transcription. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DFF ChIP-Seq, rapid viral protein degradation, and promoter mutation and function assays). In D-NT2, but not fibroblasts, RNA polymerase II initiation and elongation at several viral promoters requires viral DNA synthesis and are independent of host P-TEFb, viral IE2, or viral UL87 late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase transcription and mutations in CREB and NF-kB response elements decrease transcription. The GC-box sequence mutations also alter infected cell morphology and gene expression program, whereas mutations in CREB and NF-kB response elements do not. In D-NT2, LTF-driven promoters constitute a smaller proportion of the viral late promoter population and are generally less active. Additionally, viral genomes have more nucleosomes, potentially restricting LTF access. A TBP-IE2-nucleosome complex, with more nucleosome than in fibroblasts, covers the MIE promoter transcription start site, potentially contributing to epigenetically silence of the promoter.
2025-07-22 | GSE291635 | GEO
Project description:Cell type differences in the human cytomegalovirus (HCMV) transcriptome may result from variations in transcription and/or post-transcription. Here we report unexpected differences in transcription and epigenetic control in late-stage HCMV infection of human differentiated NTera2 cells (D-NT2) compared to fibroblasts, using integrated functional genomic approaches (PRO-Seq, RNA-Seq, DFF ChIP-Seq, rapid viral protein degradation, and promoter mutation and function assays). In D-NT2, but not fibroblasts, RNA polymerase II initiation and elongation at several viral promoters requires viral DNA synthesis and are independent of host P-TEFb, viral IE2, or viral UL87 late transcription factor (LTF). This includes transcription from the enhancer for the major immediate early (MIE) promoter where GC-box sequence mutations increase transcription and mutations in CREB and NF-kB response elements decrease transcription. The GC-box sequence mutations also alter infected cell morphology and gene expression program, whereas mutations in CREB and NF-kB response elements do not. In D-NT2, LTF-driven promoters constitute a smaller proportion of the viral late promoter population and are generally less active. Additionally, viral genomes have more nucleosomes, potentially restricting LTF access. A TBP-IE2-nucleosome complex, with more nucleosome than in fibroblasts, covers the MIE promoter transcription start site, potentially contributing to epigenetically silence of the promoter.
2025-07-22 | GSE291634 | GEO