Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1)

The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites

Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4

RNA-dependent recruitment of ORC to the Epstein -Barr virus origin of replication

The Origin Recognition Complex (ORC) plays a central role in determining the initiation sites of DNA replication in eukaryotes. In higher eukaryotes, ORC lacks sequence-specific DNA binding and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein and human TRF2. We mapped the regions of EBNA1 necessary for OriP ORC recruitment and replication to the RGG-like motifs LR1 and LR2. These regions also confer metaphase attachment ability to EBNA1. We found that LR1 and LR2 recruitment of ORC is RNA-dependent and requires a subdomain of Orc1. HMGA1a, which can functionally substitute for the RGG domains, also recruits ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs, as well as the Orc1 subdomain, bind to structured G-rich RNA. RNase A treatment of cellular chromatin released a fraction of total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We characterized the RNA responsible for EBNA1 interaction with ORC and showed that it is RNA capable of forming G-quadruplex structures. Drugs that specifically interact with G-quadruplex structures decrease EBV-positive cell viability. This may be due to the requirement of G-quadruplex structures in EBNA1-dependent replication at DS as well as metaphase attachment during cellular division. Furthermore, we showed that TRF2 interacts with the same subdomain of Orc1 as EBNA1. The recruitment of ORC by TRF2 to both OriP and telomeres is also RNA-dependent and mediated by the TERRA RNA, which is capable of forming G-quadruplexes. We propose that structural RNA molecules mediate ORC recruitment at OriP as well as some cellular origins

RNA-dependent recruitment of ORC to the Epstein -Barr virus origin of replication

The Origin Recognition Complex (ORC) plays a central role in determining the initiation sites of DNA replication in eukaryotes. In higher eukaryotes, ORC lacks sequence-specific DNA binding and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein and human TRF2. We mapped the regions of EBNA1 necessary for OriP ORC recruitment and replication to the RGG-like motifs LR1 and LR2. These regions also confer metaphase attachment ability to EBNA1. We found that LR1 and LR2 recruitment of ORC is RNA-dependent and requires a subdomain of Orc1. HMGA1a, which can functionally substitute for the RGG domains, also recruits ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs, as well as the Orc1 subdomain, bind to structured G-rich RNA. RNase A treatment of cellular chromatin released a fraction of total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We characterized the RNA responsible for EBNA1 interaction with ORC and showed that it is RNA capable of forming G-quadruplex structures. Drugs that specifically interact with G-quadruplex structures decrease EBV-positive cell viability. This may be due to the requirement of G-quadruplex structures in EBNA1-dependent replication at DS as well as metaphase attachment during cellular division. Furthermore, we showed that TRF2 interacts with the same subdomain of Orc1 as EBNA1. The recruitment of ORC by TRF2 to both OriP and telomeres is also RNA-dependent and mediated by the TERRA RNA, which is capable of forming G-quadruplexes. We propose that structural RNA molecules mediate ORC recruitment at OriP as well as some cellular origins

RNA-dependent recruitment of ORC to the Epstein -Barr virus origin of replication

The Origin Recognition Complex (ORC) plays a central role in determining the initiation sites of DNA replication in eukaryotes. In higher eukaryotes, ORC lacks sequence-specific DNA binding and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr Virus (EBV) origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein and human TRF2. We mapped the regions of EBNA1 necessary for OriP ORC recruitment and replication to the RGG-like motifs LR1 and LR2. These regions also confer metaphase attachment ability to EBNA1. We found that LR1 and LR2 recruitment of ORC is RNA-dependent and requires a subdomain of Orc1. HMGA1a, which can functionally substitute for the RGG domains, also recruits ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs, as well as the Orc1 subdomain, bind to structured G-rich RNA. RNase A treatment of cellular chromatin released a fraction of total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We characterized the RNA responsible for EBNA1 interaction with ORC and showed that it is RNA capable of forming G-quadruplex structures. Drugs that specifically interact with G-quadruplex structures decrease EBV-positive cell viability. This may be due to the requirement of G-quadruplex structures in EBNA1-dependent replication at DS as well as metaphase attachment during cellular division. Furthermore, we showed that TRF2 interacts with the same subdomain of Orc1 as EBNA1. The recruitment of ORC by TRF2 to both OriP and telomeres is also RNA-dependent and mediated by the TERRA RNA, which is capable of forming G-quadruplexes. We propose that structural RNA molecules mediate ORC recruitment at OriP as well as some cellular origins

Role for G-Quadruplex RNA Binding by Epstein-Barr Virus Nuclear Antigen 1 in DNA Replication and Metaphase Chromosome Attachment▿

Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors

Regulation of Epstein-Barr Virus Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2▿

The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G1/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability

ORC binding to TRF2 stimulates OriP replication

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein–Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits

Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1)

Abstract The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.</p