Thomas Küntziger

Reprogrammed gene expression in a somatic cell-free extract

We have developed a somatic cell‐free system that remodels chromatin and activates gene expression in heterologous differentiated nuclei. Extracts of stimulated human T cells elicit chromatin binding of transcriptional activators of the interleukin‐2 (IL‐2) gene, anchoring and activity of a chromatin‐remodeling complex and hyperacetylation of the IL‐2 promoter in purified exogenous resting T‐cell nuclei. The normally repressed IL‐2 gene is transcribed in nuclei from quiescent human T cells and from various non‐T‐cell lines. This demonstrates that somatic cell extracts can be used to reprogram gene expression in differentiated nuclei. In vitro reprogramming may be useful for investigating regulation of gene expression and for producing replacement cells for the treatment of a wide variety of diseases.

Optic atrophy 1 is an A-kinase anchoring protein on lipid droplets that mediates adrenergic control of lipolysis.

Adrenergic stimulation of adipocytes yields a cAMP signal that activates protein kinase A (PKA). PKA phosphorylates perilipin, a protein localized on the surface of lipid droplets that serves as a gatekeeper to regulate access of lipases converting stored triglycerides to free fatty acids and glycerol in a phosphorylation‐dependent manner. Here, we report a new function for optic atrophy 1 (OPA1), a protein known to regulate mitochondrial dynamics, as a dual‐specificity A‐kinase anchoring protein associated with lipid droplets. By a variety of protein interaction assays, immunoprecipitation and immunolocalization experiments, we show that OPA1 organizes a supramolecular complex containing both PKA and perilipin. Furthermore, by a combination of siRNA‐mediated knockdown, reconstitution experiments using full‐length OPA1 with or without the ability to bind PKA or truncated OPA1 fused to a lipid droplet targeting domain and cellular delivery of PKA anchoring disruptor peptides, we demonstrate that OPA1 targeting of PKA to lipid droplets is necessary for hormonal control of perilipin phosphorylation and lipolysis.

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner.

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309-691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309-691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401-->Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.

DAXX-dependent supply of soluble (H3.3–H4) dimers to PML bodies pending deposition into chromatin

Replication-independent chromatin deposition of histone variant H3.3 is mediated by several chaperones. We report a multistep targeting of newly synthesized epitope-tagged H3.3 to chromatin via PML bodies. H3.3 is recruited to PML bodies in a DAXX-dependent manner, a process facilitated by ASF1A. DAXX is required for enrichment of ATRX, but not ASF1A or HIRA, with PML. Nonetheless, the chaperones colocalize with H3.3 at PML bodies and are found in one or more complexes with PML. Both DAXX and PML are necessary to prevent accumulation of a soluble, nonincorporated pool of H3.3. H3.3 targeting to PML is enhanced with an (H3.3-H4)2 tetramerization mutant of H3.3, suggesting H3.3 recruitment to PML as an (H3.3-H4) dimer rather than as a tetramer. Our data support a model of DAXX-mediated recruitment of (H3.3-H4) dimers to PML bodies, which may function as triage centers for H3.3 deposition into chromatin by distinct chaperones.

Chromatin Environment of Histone Variant H3.3 Revealed by Quantitative Imaging and Genome-scale Chromatin and DNA Immunoprecipitation

Histone variant H3.3 is loaded onto chromatin in a replication-independent manner, but the epigenetic environment of H3.3 is unclear. Quantitative imaging and chromatin immunoprecipitation show that in mesenchymal stem cells H3.3 targets lineage-priming genes with a potential for activation facilitated by a permissive chromatin environment.

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response

The function of protein phosphatase 1 nuclear‐targeting subunit (PNUTS)—one of the most abundant nuclear‐targeting subunits of protein phosphatase 1 (PP1c)—remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing‐radiation‐induced DNA damage. Overexpression of PNUTS–enhanced green fluorescent protein (EGFP)—which is rapidly and transiently recruited at DNA damage sites—inhibits G2 arrest. Finally, γH2AX, p53‐binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS‐depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.

Protein phosphatase 1 regulators in DNA damage signaling

In response to DNA damaging agents and endogenous DNA lesions, human cells activate signaling cascades and repair mechanisms to help maintain genomic integrity. Phosphorylation plays a major role in DNA damage signaling, and the role of Ser/Thr kinases, including ATM, ATR, CHK1, CHK2 and DNA-PK, is particularly well documented. While these kinases have taken the center stage in DNA damage signaling until now, a role for Ser/Thr phosphatases is emerging, including Protein Phosphatase 1 (PP1). PP1 substrate specificity is regulated by its binding to a large number of different targeting subunits, and several of these have recently been identified as regulators of DNA damage responses. Here we review recent progress regarding the involvement of PP1 and its binding partners in DNA damage signaling.

Mutually exclusive binding of PP1 and RNA to AKAP149 affects the mitochondrial network

A-kinase-anchoring protein 149 (AKAP149) is a membrane protein of the mitochondrial and endoplasmic reticulum/nuclear envelope network. AKAP149 controls the subcellular localization and temporal order of protein phosphorylation by tethering protein kinases and phosphatases to these compartments. AKAP149 also includes an RNA-binding K homology (KH) domain, the loss of function of which has been associated in other proteins with neurodegenerative syndromes. We show here that protein phosphatase 1 (PP1) binding occurs through a conserved RVXF motif found in the KH domain of AKAP149 and that PP1 and RNA binding to this same site is mutually exclusive and controlled through a novel, phosphorylation-dependent mechanism. A collapse of the mitochondrial network is observed upon introduction of RNA-binding deficient mutants of AKAP149, pointing to the importance of RNA tethering to the mitochondrial membrane by AKAP149 for mitochondrial distribution.

Distinct kinetics of DNA repair protein accumulation at DNA lesions and cell cycle‐dependent formation of γH2AX‐ and NBS1‐positive repair foci

The DNA damage response is a fundamental, well‐regulated process that occurs in the genome to recognise DNA lesions. Here, we studied kinetics of proteins involved in DNA repair pathways and their recruitment to DNA lesions during the cell cycle. In non‐irradiated and irradiated cells, we analysed the distribution pattern and spatiotemporal dynamics of γH2AX, 53BP1, BMI1, MDC1, NBS1, PCNA, coilin and BRCA1 proteins.

Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters, but does not replicate in, mammalian cells.

BackgroundThe aquatic birnavirus infectious pancreatic necrosis virus (IPNV) causes infectious pancreatic necrosis (IPN), a severe disease in farmed salmonid fish. IPNV has a very broad host range and infects many different species of fish as well as molluscs and crustaceans. Investigation of the host reservoir of a virus may reveal important molecular mechanisms governing the infection processes such as receptors and entry mechanisms. In the present work we have studied whether IPNV is able to infect cells with different mammalian origin.ResultsIPNV bound in a specific manner to a membrane protein of the rabbit kidney cell line RK-13 as shown by the use of a virus overlay protein binding assay (VOPBA). Six different mammalian cell lines were inoculated with IPNV and incubated in parallels at different temperatures. At 7 days post inoculation (dpi), IPNV was detected by indirect immunofluorescent antibody test (IFAT) in all the cell lines. Confocal microscopy confirmed intracellular presence of the virus. No apparent cytopathic effect (cpe) was observed in any of the cultures, and no viral replication was demonstrated with real-time RT-PCR.ConclusionOur results show that IPNV is able to enter into a wide range of mammalian cells, and virus entry is most likely receptor mediated. We found no indication of IPNV replication in any of the mammalian cell lines tested.