Aloys Schepers

Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein–Barr virus

Epstein–Barr virus (EBV) replicates in its latent phase once per cell cycle in proliferating B cells. The latent origin of DNA replication, oriP, supports replication and stable maintenance of the EBV genome. OriP comprises two essential elements: the dyad symmetry (DS) and the family of repeats (FR), both containing clusters of binding sites for the transactivator EBNA1. The DS element appears to be the functional replicator. It is not yet understood how oriP‐dependent replication is integrated into the cell cycle and how EBNA1 acts at the molecular level. Using chromatin immunoprecipitation experiments, we show that the human origin recognition complex (hsORC) binds at or near the DS element. The association of hsORC with oriP depends on the DS element. Deletion of this element not only abolishes hsORC binding but also reduces replication initiation at oriP to background level. Co‐immunoprecipitation experiments indicate that EBNA1 is associated with hsORC in vivo. These results indicate that oriP might use the same cellular initiation factors that regulate chromosomal replication, and that EBNA1 may be involved in recruiting hsORC to oriP.

The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold

The Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the earliest viral proteins expressed after infection and is the only latent protein consistently expressed in viral-associated tumors. EBNA1s crucial role in viral DNA replication, episomal maintenance, and partitioning is well examined whereas its importance for the immortalization process and the tumorgenicity of EBV is unclear. To address these open questions, we generated, based on the maxi-EBV system, an EBNA1-deficient EBV mutant and used this strain to infect primary human B cells. Surprisingly, lymphoblastoid cell lines (LCL) emerged from these experiments, although with very low frequency. These cell lines were indistinguishable from normal LCLs with respect to proliferation and growth conditions. A detailed analysis indicated that the entire viral DNA was integrated into the cellular genome. At least 5 of the 11 latent EBV proteins were expressed, indicating the integrity of the EBV genome. EBNA1-positive and ΔEBNA1-EBV-LCLs were injected into severe combined immunodeficient (SCID) mice to examine their tumorgenicity in comparison. Both groups supported tumor growth, indicating that EBNA1 is not mandatory for EBVs oncogenic potential. The results shown provide genetic evidence that EBNA1 is not essential to establish LCLs but promotes the efficiency of this process significantly.

CD8 T Cell Recognition of Endogenously Expressed Epstein-Barr Virus Nuclear Antigen 1

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 contains a glycine-alanine repeat (GAr) domain that appears to protect the antigen from proteasomal breakdown and, as measured in cytotoxicity assays, from major histocompatibility complex (MHC) class I–restricted presentation to CD8+ T cells. This led to the concept of EBNA1 as an immunologically silent protein that although unique in being expressed in all EBV malignancies, could not be exploited as a CD8 target. Here, using CD8+ T cell clones to native EBNA1 epitopes upstream and downstream of the GAr domain and assaying recognition by interferon γ release, we show that the EBNA1 naturally expressed in EBV-transformed lymphoblastoid cell lines (LCLs) is in fact presented to CD8+ T cells via a proteasome/peptide transporter–dependent pathway. Furthermore, LCL recognition by such CD8+ T cells, although slightly lower than seen with paired lines expressing a GAr-deleted EBNA1 protein, leads to strong and specific inhibition of LCL outgrowth in vitro. Endogenously expressed EBNA1 is therefore accessible to the MHC class I pathway despite GAr-mediated stabilization of the mature protein. We infer that EBNA1-specific CD8+ T cells do play a role in control of EBV infection in vivo and might be exploitable in the control of EBV+ malignancies.

Cell cycle regulation of chromatin at an origin of DNA replication.

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein–Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle‐dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS‐bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS‐bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP‐dependent chromatin‐remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1‐specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h–HDAC1/2 complex coordinates G1‐specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.

Complex protein-DNA dynamics at the latent origin of DNA replication of Epstein-Barr virus.

The sequential binding of the origin recognition complex (ORC), Cdc6p and the minichromosome maintenance proteins (MCM2-7) mediates replication competence at eukaryotic origins of DNA replication. The latent origin of Epstein-Barr virus, oriP, is a viral origin known to recruit ORC. OriP also binds EBNA1, a virally encoded protein that lacks any activity predicted to be required for replication initiation. Here, we used chromatin immunoprecipitation and chromatin binding to compare the cell-cycle-dependent binding of pre-RC components and EBNA1 to oriP and to global cellular chromatin. Prereplicative-complex components such as the Mcm2p-Mcm7p proteins and HsOrc1p are regulated in a cell-cycle-dependent fashion, whereas other HsOrc subunits and EBNA1 remain constantly bound. In addition, HsOrc1p becomes sensitive to the 26S proteasome after release from DNA during S phase. These results show that the complex protein-DNA dynamics at the viral oriP are synchronized with the cell division cycle. Chromatin-binding and chromatin-immunoprecipitation experiments on G0 arrested cells indicated that the ORC core complex (ORC2-5) and EBNA1 remain bound to chromatin and oriP. HsOrc6p and the MCM2-7 complex are released in resting cells. HsOrc1p is partly liberated from chromatin. Our data suggest that origins remain marked in resting cells by the ORC core complex to ensure a rapid and regulated reentry into the cell cycle. These findings indicate that HsOrc is a dynamic complex and that its DNA binding activity is regulated differently in the various stages of the cell cycle.

Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins

In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G1 and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.

RNA‐dependent recruitment of the origin recognition complex

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. 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 origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus‐encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG‐like motifs, referred to as LR1 and LR2, in the EBNA1 amino‐terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA‐dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G‐rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

Eri1 degrades the stem-loop of oligouridylated histone mRNAs to induce replication-dependent decay

The exoRNase Eri1 inhibits RNA interference and trims the 5.8S rRNA 3′ end. It also binds to the stem-loop of histone mRNAs, but the functional importance of this interaction remains elusive. Histone mRNAs are normally degraded at the end of S phase or after pharmacological inhibition of replication. Both processes are impaired in Eri1-deficient mouse cells, which instead accumulate oligouridylated histone mRNAs. Eri1 trims the mature histone mRNAs by two unpaired nucleotides at the 3′ end but stalls close to the double-stranded stem. Upon oligouridylation of the histone mRNA, the Lsm1–7 heteroheptamer recognizes the oligo(U) tail and interacts with Eri1, whose catalytic activity is then able to degrade the stem-loop in a stepwise manner. These data demonstrate how degradation of histone mRNAs is initiated when 3′ oligouridylation creates a cis element that enables Eri1 to process the double-stranded stem-loop structure.

Differential Binding of Replication Proteins across the Human c-myc Replicator

ABSTRACT The binding of the prereplication complex proteins Orc1, Orc2, Mcm3, Mcm7, and Cdc6 and the novel DNA unwinding element (DUE) binding protein DUE-B to the endogenous human c-myc replicator was studied by chromatin immunoprecipitation. In G1-arrested HeLa cells, Mcm3, Mcm7, and DUE-B were prominent near the DUE, while Orc1 and Orc2 were least abundant near the DUE and more abundant at flanking sites. Cdc6 binding mirrored that of Orc2 in G1-arrested cells but decreased in asynchronous or M-phase cells. Similarly, the signals from Orc1, Mcm3, and Mcm7 were at background levels in cells arrested in M phase, whereas Orc2 retained the distribution seen in G1-phase cells. Previously shown to cause histone hyperacetylation and delocalization of replication initiation, trichostatin A treatment of cells led to a parallel qualitative change in the distribution of Mcm3, but not Orc2, across the c-myc replicator. Orc2, Mcm3, and DUE-B were also bound at an ectopic c-myc replicator, where deletion of sequences essential for origin activity was associated with the loss of DUE-B binding or the alteration of chromatin structure and loss of Mcm3 binding. These results show that proteins implicated in replication initiation are selectively and differentially bound across the c-myc replicator, dependent on discrete structural elements in DNA or chromatin.

Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay

ABSTRACT Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr virus latent origin oriP. oriP contains two elements, an origin (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G1 phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.