Dataset Information

ChIP-seq targeting Myc-tagged UL34 from human fibroblasts infected with human cytomegalovirus at 48 hours post infection

ABSTRACT: Purpose: To identify interactions of the viral DNA-binding protein, UL34, with the viral genome during lytic-phase replication. Methods: Human fibroblasts were infected with human cytomegalovirus expressing Myc-tagged UL34 in biological duplicate. At 48 hours post infection, ChIP was performed with anti-Myc tag antibody (Cell Signaling) and the resulting DNA fragments were sequenced on Illumina MiSeq. Reads were trimmed and duplicates removed. Reads passing quality filter were aligned to a custom reference genome containing both the human genome with the human cytomegalovirus genome as an additional chromosome to prevent read assignment bias with CLC Genomics Workbench verison 9.0. Enrichment of UL34 in the viral origin of lytic replication (oriLyt) was calculated by dividing read counts from the ChIP'd samples (Myc-UL34-1, Myc-UL34-2) by those from non-ChIP whole cell lysate (Input) with a 125bp sliding window (25bp overhang) after adjusting for total read count variances. An additional sample containing DNA that was subjected to the ChIP protocol in the absence of antibody (No Antibody) was compared to the Input to determine background enrichment. Results: Several peaks were identified when comparing the Myc-UL34-ChIP samples to Input. UL34 is bound to the viral oriLyt at 48 hours post infection near the three previously defined UL34-binding sites to varying degrees, as well as additional locations within the region. No peaks were identified when comparing the No Antibody and Input samples.

ORGANISM(S): Homo sapiens

PROVIDER: GSE106211 | GEO | 2018/05/17

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:The Epstein-Barr virus (EBV) switches between latent and lytic phases in hosts that are important in developing related diseases. However, the underlying mechanism of how the viral latent-lytic switch is controlled and how EBV itself mediates this regulation remains largely unknown. This study identified the upregulated histone acetyl reader bromodomain-containing protein 7 (BRD7) during EBV latent infection. Based on the ChIP-sequencing of endogenous BRD7 in Burkitt lymphoma cells, we found that EBV drove BRD7 to regulate cellular and viral genomic loci, including the transcriptional activation of c-Myc, a recently reported regulator of EBV latency. Additionally, EBV-mediated BRD7 signals enriched around the FUSE site in chromosome 8, and the enhancer LOC108348026 in the lgH locus, which might activate the c-Myc alleles. Mechanically, EBV-encoded nuclear antigen 1 (EBNA1) bound and recruited BRD7 to colocalize at promotor regions of the related genes, thus serving as cofactors for the maintenance of viral latency. Moreover, the disruption of BRD7 decreased the c-Myc expression, induced the BZLF1 expression, and reactivated the lytic cycle. Our findings reveal the unique role of BRD7 hijacked by EBV in maintaining the viral latency state via chromatin remodeling. The study paved the way for understanding the new molecular mechanism of EBV-induced chromatin remodeling and latent-lytic switch regulation, providing novel therapeutic candidate targets for EBV persistent infection. With establishing persistent infection in most human hosts, EBV is usually at a latent infection state. How the viral latency is maintained in cells remains largely unknown. c-Myc was recently reported to act as a controller of the lytic switch, while whether and how EBV regulates it remains to be explored. Here, we identified that BRD7 is involved in controlling EBV latency. We found that EBV-mediated BRD7 was enriched in both the normal promoter regions and the translocation alleles of c-Myc, and the disruption of BRD7 decreased c-Myc expression to reactivate the lytic cycle. We also demonstrated that EBV-encoded EBNA1 bound to and regulated BRD7. Therefore, we reveal a novel mechanism by which EBV can regulate its infection state by hijacking host BRD7 to target c-Myc. Our findings will help future therapeutic intervention strategies of EBV infection and pathogenesis.

Project description:Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes defcient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-defcient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function. [i] H3 ChIP-chip measurements of WT human cytomegalovirus (strain TB40E) following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding mock IgG input control: 3 replicates; WT virus, 48 hours postinfection with corresponding mock IgG input contro: 3 replicates. [ii] MNase-chip measurements of WT and dlIE1 human cytomegalovirus nucleosomes following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT virus 48 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 8 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates. [iii] Total and nascent RNA measurements of WT and dlIE1 human cytomegalovirus transcripts following infection of MRC-5 cells. WT 8 hours postinfection: 2 replicates; WT virus 96 hours postinfection: 2 replicates; dlIE1 8 hours postinfection: 2 replicates; dlIE1 virus 96 hours postinfection: 2 replicates; sonicated DNA input control: 10 replicates.

Project description:Enhancers play indispensable roles in cell proliferation and survival through spatiotemporally regulating gene transcription. In addition, active enhancers and super-enhancers often produce noncoding enhancer RNAs (eRNAs) that precisely control RNA polymerase II activity. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human oncogenic gamma-2 herpesvirus that causes Kaposi’s sarcoma and lymphoproliferative diseases of B-cell origin such as primary effusion lymphoma (PEL). It is well characterized that KSHV utilizes host epigenetic and nuclear machineries to control the switch between two life cycles, latency and lytic replication. However, how KSHV impacts the host epigenome at different stages of viral life cycle is not well understood. Using the analysis of global run-on sequencing (GRO-seq) and chromatin-immunoprecipitation sequencing (ChIP-seq), we profiled the dynamics of host transcriptional regulatory elements during latency and lytic replication of KSHV-infected PEL cells. This study showed that a number of critical host genes for KSHV latency, including MYC proto-oncogene, were under the control of super-enhancers and eRNAs that were globally repressed upon viral reactivation. A combination of circular chromosome conformation capture combined with sequencing (4C-seq), GRO-seq and ChIP-seq indicated that the eRNA-expressing super-enhance regions were located at downstream of the MYC gene in KSHV-infected PELs. Treatment of an epigenetic drug to block enhancer function or shRNA-mediated depletion of the eRNA expression significantly reduced MYC mRNA expression in KSHV-infected PELs. Finally, while cellular IRF4 acted upon the eRNAs and super-enhancers for MYC expression during latency, the KSHV viral IRF4 repressed cellular IRF4 expression upon reactivation, decreasing MYC expression and thereby, facilitating lytic replication. Taken together, these data suggest that KSHV acts as an epigenetic driver that modifies host epigenomic status by effectively regulating enhancer function upon reactivation.

Dataset Information

ChIP-seq targeting Myc-tagged UL34 from human fibroblasts infected with human cytomegalovirus at 48 hours post infection

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets