No abstract

Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the viral protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell generation after removal of selection (A. L. Kirchmaier and B. Sugden, J. Virol. 69:1280-1283, 1995; B. Sugden and N. Warren, Mol. Biol. Med. 5:85-94, 1988). We refer to these plasmids as established replicons in that they support efficient DNA synthesis and partitioning each cell cycle. Unexpectedly, we have found that upon introduction of oriP plasmids into a population of EBNA-1-positive cells, oriP plasmids replicate but are lost precipitously from cells during 2 weeks posttransfection (>25% rate of loss per cell generation). Upon investigation of these disparate observations, we have found that only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA-1 and hygromycin phosphotransferase give rise to drug-resistant clones in which the oriP replicon is established. A hereditable alteration in these drug-resistant cell clones, manifested at the genetic or epigenetic level, does not underlie the establishment of oriP, as newly introduced oriP plasmids replicate but are also lost rapidly from these cells. In addition, a genetic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escherichia coli, and reintroduced into EBNA-1-positive cells are likewise established inefficiently. Our findings demonstrate that oriP replicons are not intrinsically stable in EBNA-1-positive cell lines. Rather, the establishment of an oriP replicon is conferred upon the replicon by a stochastic, epigenetic event that occurs infrequently and, therefore, is detected in only a minority of cells.It is essential for cells and latently infecting viruses to duplicate their genomes and transmit their genetic information to daughter cells during each cell cycle, processes that we condense into the term replication. The plasmid replicon of Epstein-Barr virus (EBV) serves as a model for studying replication in that, akin to the genome of its host cell, this replicon is synthesized only once per cell cycle during S phase (1, 51) and is efficiently partitioned to daughter cells (20,25,45,47). Only two viral components are required for the replication of EBV's genome, the latent origin oriP and its binding protein EpsteinBarr-associated nuclear antigen 1 (EBNA-1); all else is contributed by the cell (26, 49, 52). Plasmids containing oriP support efficient replication in EBNA-1-positive cells selected to maintain them, being lost at a rate of 2 to 4% per cell generation after removal of selection (20, 47), a rate of loss which resembles that of ARS/CEN plasmids in Saccharomyces cerevisiae (23). We define these plasmids as established replicons in that they support efficient DNA synthesis and partitioning each cell cycle.What events ensure efficient replication of oriP plasmids? The answer to this question largely remains enigmatic...

The LIN-1 ETS transcription factor inhibits vulval cell fates during Caenorhabditis elegans development. We demonstrate that LIN-1 interacts with UBC-9, a small ubiquitin-related modifier (SUMO) conjugating enzyme. This interaction is mediated by two consensus sumoylation motifs in LIN-1. Biochemical studies showed that LIN-1 is covalently modified by SUMO-1. ubc-9 and smo-1, the gene encoding SUMO-1, inhibit vulval cell fates and function at the level of lin-1, indicating that sumoylation promotes LIN-1 inhibition of vulval cell fates. Sumoylation of LIN-1 promoted transcriptional repression and mediated an interaction with MEP-1, a protein previously shown to associate with the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies showed that mep-1 inhibits vulval cell fates and functions at the level of lin-1. We propose that sumoylation of LIN-1 mediates an interaction with MEP-1 that contributes to transcriptional repression of genes that promote vulval cell fates. These studies identify a molecular mechanism for SUMO-mediated transcriptional repression.

We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a−/− bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a−/− hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a−/− mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic “state” created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.