Deletion of PDK1 Causes Cardiac Sodium Current Reduction in Mice

Background

The AGC protein kinase family regulates multiple cellular functions. 3-phosphoinositide-dependent protein kinase-1 (PDK1) is involved in the pathogenesis of arrhythmia, and its downstream factor, Forkhead box O1 (Foxo1), negatively regulates the expression of the cardiac sodium channel, Nav1.5. Mice are known to die suddenly after PDK1 deletion within 11 weeks, but the underlying electrophysiological bases are unclear. Thus, the aim of this study was to investigate the potential mechanisms between PDK1 signaling pathway and cardiac sodium current.

Methods and Results

Using patch clamp and western blotting techniques, we investigated the role of the PDK1-Foxo1 pathway in PDK1 knockout mice and cultured cardiomyocytes. We found that PDK1 knockout mice undergo slower heart rate, prolonged QRS and QTc intervals and abnormal conduction within the first few weeks of birth. Furthermore, the peak sodium current is decreased by 33% in cells lacking PDK1. The phosphorylation of Akt (308T) and Foxo1 (24T) and the expression of Nav1.5 in the myocardium of PDK1-knockout mice are decreased, while the nuclear localization of Foxo1 is increased. The role of the PDK1-Foxo1 pathway in regulating Nav1.5 levels and sodium current density was verified using selective PDK1, Akt and Foxo1 inhibitors and isolated neonatal rat cardiomyocytes.

Conclusion

These results indicate that PDK1 participates in the dysregulation of electrophysiological basis by regulating the PDK1-Foxo1 pathway, which in turn regulates the expression of Nav1.5 and cardiac sodium channel function.     

Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition,bi-directionally from the primed protospacer

Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼350 new spacers acquired in priming events and identified a 5′-protospacer-GG-3′ protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2–3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.     

Inducible chromosomal translocation of AML1 and ETO genes through Cre/loxP-mediated recombination in the mouse

Transgenic mice have been used to explore the role of chromosomal translocations in the genesis of tumors. But none of these efforts has actually involved induction of a translocation in vivo. Here we report the use of Cre recombinase to replicate in vivo the t(8;21) translocation found in human acute myeloid leukemia (AML). As in the human tumors, the murine translocation fuses the genes AML1 and ETO. We used homologous recombination to place loxP sites at loci that were syntenic with the break points for the human translocation. Cre activity was provided in mice by a transgene under the control of the Nestin promoter, or in cultured B cells by infecting with a retroviral vector encoding Cre. In both instances, Cre activity mediated interchromosomal translocations that fused the AML1 and ETO genes. Thus, reciprocal chromosomal translocations that closely resemble rearrangements found in human cancers can be achieved in mice.     

Silencing Pax3 by shRNA inhibits the proliferation and differentiation of duck (Anas platyrhynchos) myoblasts

The Pax3 gene has been proven to play a crucial role in determining myogenic progenitor cell fate during embryonic myogenesis; however, the molecular role of Pax3 in myoblast development during later stages of myogenesis is unknown. We hypothesized that Pax3 would function in myoblast proliferation and differentiation; therefore, we employed three short hairpin RNAs (shRNAs) (shRNA1, shRNA2, and shRNA3) that target Pax3 to characterize the function of Pax3 in duck myoblast development. The mRNA and protein expression levels of Pax3 in duck myoblasts were detected using real-time PCR and Western blotting. Cell proliferation was assessed using the MTT and BrdU assays, while cell differentiation was assayed using immunofluorescence labeling with a MyoG antibody. Additionally, folic acid (FA), which is a rescue tool, was added into the medium of duck myoblasts to indirectly examine the function of Pax3 on duck myoblast proliferation and differentiation. The results revealed that one of the shRNA vectors, shRNA1, could significantly and stably reduce the expression of Pax3 (P < 0.05). Silencing Pax3 by shRNA1 significantly reduced the proliferation and differentiation of duck myoblasts (P < 0.05) due to downregulated expression of myogenic regulator factors. These trends could be rescued by adding FA; and Pax7, a paralog gene of Pax3, was involved in those processes. Overall, Pax3 had a positive function in duck myoblast proliferation and differentiation by modulating the expression of myogenic regulation factors, and shRNA targeting of Pax3 might be a new approach for understanding the function of Pax3 in the development of diverse tissues.     

Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression

Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5′ portion derives from S. cerevisiae and the 3′ portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not require repeated backcrossing to one of the parental species.     

Synteny mapping in the bovine: genes from human chromosome 4.

Genes homologous to those located on human chromosome 4 (HSA4) were mapped in the bovine to determine regions of syntenic conservation among humans, mice, and cattle. Previous studies have shown that two homologs of genes on HSA4, PGM2 and PEPS, are located in bovine syntenic group U15 (chromosome 6). The homologous mouse genes, Pgm-1 and Pep-7, are on MMU5. Using a panel of bovine x hamster hybrid somatic cells, we have assigned homologs of 11 additional HSA4 loci to their respective bovine syntenic groups. D4S43, D4S10, QDPR, IGJ, ADH2, KIT, and IF were assigned to syntenic group U15. This syntenic arrangement is not conserved in the mouse, where D4s43, D4s10, Qdpr, and Igj are on MMU5 while Adh-2 is on MMU3. IL-2, FGB, FGG, and F11, which also reside on MMU3, were assigned to bovine syntenic group U23. These data suggest that breaks and/or fusions of ancestral chromosomes carrying these genes occurred at different places during the evolution of humans, cattle, and mice.     

Tcrδ translocations that delete the Bcl11b haploinsufficient tumor suppressor gene promote atm-deficient T cell acute lymphoblastic leukemia

ATM is the master regulator of the cellular response to DNA double strand breaks (DSBs). Deficiency of ATM predisposes humans and mice to αβ T lymphoid cancers with clonal translocations between the T cell receptor (TCR) α/δ locus and a 450 kb region of synteny on human chromosome 14 and mouse chromosome 12. While these translocations target and activate the TCL1 oncogene at 14q32 to cause T cell pro-lymphocytic leukemia (T-PLL), the TCRα/δ;14q32 translocations in ATM-deficient T cell acute lymphoblastic leukemia (T-ALL) have not been characterized and their role in cancer pathogenesis remains unknown. The corresponding lesion in Atm-deficient mouse T-ALLs is a chromosome t(12;14) translocation with Tcrδ genes fused to sequences on chromosome 12; although these translocations do not activate Tcl1, they delete the Bcl11b haploinsufficient tumor suppressor gene. To assess whether Tcrδ translocations that inactivate one copy of Bcl11b promote transformation of Atm-deficient cells, we analyzed Atm^−/− mice with mono-allelic Bcl11b deletion initiating in thymocytes concomitant with Tcrδ recombination. Inactivation of one Bcl11b copy had no effect on the predisposition of Atm^−/− mice to clonal T-ALLs. Yet, none of these T-ALLs had a clonal chromosome t(12;14) translocation that deleted Bcl11b indicating that Tcrδ translocations that inactivate a copy of Bcl11b promote transformation of Atm-deficient thymocytes. Our data demonstrate that antigen receptor locus translocations can cause cancer by deleting a tumor suppressor gene. We discuss the implications of these findings for the etiology and therapy of T-ALLs associated with ATM deficiency and TCRα/δ translocations targeting the 14q32 cytogenetic region.     

Reciprocal Translocations and Translocation Disomics of Aspergillus and Their Use for Genetic Mapping

Two new techniques are described for genetic mapping of reciprocal translocations in A. nidulans, which can be used to locate centromeres and meiotically unlinked markers. They both make use of unbalanced disomics from heterozygous translocation crosses. These are mainly hyperhaploids of two classes: either typical-looking n + 1 with a normal chromosome in addition to a haploid set containing the translocation, or translocation disomics. When large chromosome segments are involved, such disomics, as well as stable aneuploids and duplication types, show characteristic phenotypes and can be classified visually. The first method maps translocation breaks qualitatively, since translocated markers can be identified when translocation disomics are analyzed for heterozygous markers. The second method measures meiotic linkage of any marker to the translocation breaks when allele ratios in the balanced haploid sectors of either or both classes of disomics are determined: linked markers show reciprocal deviations from 1:1—In addition, it can be shown that frequencies of nondisjunction and recovery of specific translocation disomics both depend on the relative position of the break within a chromosome arm. Such information can provide a rough estimate of the positions of breaks for a new translocation.—Using these techniques, as well as mitotic mapping in homo- and heterozygous translocation diploids, four reciprocal translocations were mapped. From these results, information on the sequence and orientation of most of the "meiotic fragments" of the current maps (groups III, VI, VII and VIII) was obtained, and the position of the centromeres of groups VI and VII were identified. Translocation disomics are also used to map meiotically unlinked single genes, e.g. oliA of group VII, to specify chromosome segments.     

Mechanism of Suppression of Chromosomal Instability by DNA Polymerase POLQ

Although a defect in the DNA polymerase POLQ leads to ionizing radiation sensitivity in mammalian cells, the relevant enzymatic pathway has not been identified. Here we define the specific mechanism by which POLQ restricts harmful DNA instability. Our experiments show that Polq-null murine cells are selectively hypersensitive to DNA strand breaking agents, and that damage resistance requires the DNA polymerase activity of POLQ. Using a DNA break end joining assay in cells, we monitored repair of DNA ends with long 3′ single-stranded overhangs. End joining events retaining much of the overhang were dependent on POLQ, and independent of Ku70. To analyze the repair function in more detail, we examined immunoglobulin class switch joining between DNA segments in antibody genes. POLQ participates in end joining of a DNA break during immunoglobulin class-switching, producing insertions of base pairs at the joins with homology to IgH switch-region sequences. Biochemical experiments with purified human POLQ protein revealed the mechanism generating the insertions during DNA end joining, relying on the unique ability of POLQ to extend DNA from minimally paired primers. DNA breaks at the IgH locus can sometimes join with breaks in Myc, creating a chromosome translocation. We found a marked increase in Myc/IgH translocations in Polq-defective mice, showing that POLQ suppresses genomic instability and genome rearrangements originating at DNA double-strand breaks. This work clearly defines a role and mechanism for mammalian POLQ in an alternative end joining pathway that suppresses the formation of chromosomal translocations. Our findings depart from the prevailing view that alternative end joining processes are generically translocation-prone.