During latency, Epstein-Barr virus (EBV) is stably maintained as a circular plasmid that is replicated once per cell cycle and partitioned at mitosis. Both these processes require a single viral protein, EBV nuclear antigen 1 (EBNA1), which binds two clusters of cognate binding sites within the latent viral origin, oriP. EBNA1 is known to associate with cellular metaphase chromosomes through chromosome-binding domains within its amino terminus, an association that we have determined to be required not only for the partitioning of oriP plasmids but also for their replication. One of the chromosome-binding domains of EBNA1 associates with a cellular nucleolar protein, EBP2, and it has been proposed that this interaction underlies that ability of EBNA1 to bind metaphase chromosomes. Here we demonstrate that EBNA1's chromosome-binding domains are AT hooks, a DNA-binding motif found in a family of proteins that bind the scaffold-associated regions on metaphase chromosomes. Further, we demonstrate that the ability of EBNA1 to stably replicate and partition oriP plasmids correlates with its AT hook activity and not its association with EBP2. Finally, we examine the contributions of EBP2 toward the ability of EBNA1 to associate with metaphase chromosomes in human cells, as well as support the replication and partitioning of oriP plasmids in human cells. Our results indicate that it is unlikely that EBP2 directly mediates these activities of EBNA1 in human cells.The latent Epstein-Barr virus (EBV) genome is stably retained in cells extrachromosomally as a circular plasmid. Stable replication of the EBV genome relies on the latently expressed viral protein EBV nuclear antigen 1 (EBNA1) binding to both the family of repeats (FR) within oriP, as well as to cellular chromosomes. This tethering of viral plasmids to cellular chromosomes is hypothesized to be necessary for their replication during S phase and partitioning during mitosis.Homodimers of EBNA1 are bound via a carboxy-terminal DNA-binding domain (DBD; amino acids [aa] 451 to 640) to two clusters of binding sites within oriP (60) called the FR and the dyad symmetry element (DS), whereas the amino terminus of EBNA1 (aa 1 to 450) is required for an association with cellular chromosomes (37, 48). During S phase, in concert with cellular chromosomes, oriP-plasmids are semiconservatively replicated (1, 2, 11, 47, 61), a process mediated by EBNA1's binding at the DS (7). However, the DBD of EBNA1 is by itself insufficient to recruit the licensed cellular replication apparatus to DS (28) but requires that the amino terminus of EBNA1 contain domain(s) that associate with mitotic chromosomes (48). We have recently demonstrated that the entire amino terminus of EBNA1 can be replaced by a cellular protein that specifically associates with mitotic chromosomes and that such a fusion supports the stable replication and partitioning of oriP plasmids similarly to wild-type EBNA1. In contrast, a fusion in which the amino terminus associates with interphase chromatin, but not mitotic ...

The mouse Y chromosome long arm (Yq) comprises approximately 70 Mb of repetitive, male-specific DNA together with a short (0.7-Mb) pseudoautosomal region (PAR). The repetitive non-PAR region (NPYq) encodes genes whose deficiency leads to subfertility and infertility, resulting from impaired spermiogenesis. In XSxr(a)Y*(X) mice, the only Y-specific material is provided by the Y chromosome short arm-derived sex reversal factor Sxr(a), which is attached to the X chromosome PAR; these males (NPYq- males) produce sperm with severely malformed heads and are infertile. In the present study, we investigated sperm function in these mice in the context of intracytoplasmic sperm injection (ICSI). Of 261 oocytes injected, 103 reached the 2-cell stage, and 46 developed to liveborn offspring. Using Xist RT-PCR genotyping as well as gamete and somatic cell karyotyping, all six predicted genotypes were identified among ICSI-derived progeny. The sex chromosome constitution of NPYq- males does not allow production of offspring with the same genotype, but one of the expected offspring genotypes is XY*(X)Sxr(a) (NPYq-(2)), which has the same Y gene complement as NPYq-. Analysis of NPYq-(2) males revealed they had normal-sized testes with ongoing spermatogenesis. Like NPYq- males, these males were infertile, and their sperm had malformed heads that nevertheless fertilized eggs via ICSI. In vitro fertilization (IVF), however, was unsuccessful. Overall, we demonstrated that a lack of NPYq-encoded genes does not interfere with the ability of sperm to fertilize oocytes via ICSI but does prevent fertilization via IVF. Thus, NPYq-encoded gene functions are not required after the sperm have entered the oocyte. The present work also led to development of a new mouse model lacking NPYq gene complement that will facilitate future studies of Y-encoded gene function.

Rapid alterations in cellular metabolism allow tissues to maintain homeostasis during changes in energy availability. The central metabolic regulator acetyl-CoA carboxylase 2 (ACC2) is robustly phosphorylated during cellular energy stress by AMP-activated protein kinase (AMPK) to relieve its suppression of fat oxidation. While ACC2 can also be hydroxylated by prolyl hydroxylase 3 (PHD3), the physiological consequence thereof is poorly understood. We find that ACC2 phosphorylation and hydroxylation occur in an inverse fashion. ACC2 hydroxylation occurs in conditions of high energy and represses fatty acid oxidation. PHD3-null mice demonstrate loss of ACC2 hydroxylation in heart and skeletal muscle and display elevated fatty acid oxidation. Whole body or skeletal muscle-specific PHD3 loss enhances exercise capacity during an endurance exercise challenge. In sum, these data identify an unexpected link between AMPK and PHD3, and a role for PHD3 in acute exercise endurance capacity and skeletal muscle metabolism.

Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD–HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD–HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [ 14 C]O 2 release from labeled [ 14 C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC 50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.