Kirsten Jensen

PML protein isoforms and the RBCC/TRIM motif

PML is a component of a multiprotein complex, termed nuclear bodies, and the PML protein was originally discovered in patients suffering from acute promyelocytic leukaemia (APL). APL is associated with a reciprocal chromosomal translocation of chromosomes 15 and 17, which results in a fusion protein comprising PML and the retinoic acid receptor α. The PML genomic locus is approximately 35 kb and is subdivided into nine exons. A large number of alternative spliced transcripts are synthesized from the PML gene, resulting in a variety of PML proteins ranging in molecular weight from 48–97 kDa. In this review we summarize the data on the known PML isoforms and splice variants and present a new unifying nomenclature. Although, the function/s of the PML variants are unclear, all PML isoforms contain an identical N-terminal region, suggesting that these sequences are indispensable for function, but differ in their C-terminal sequences. The N-terminal region harbours a RING-finger, two B-boxes and a predicted α-helical Coiled-Coil domain, that together form the RBCC/TRIM motif found in a large family of proteins. In PML this motif is essential for PML nuclear body formation in vivo and PML-homo and hetero interactions conferring growth suppressor, apoptotic and anti-viral activities. In APL oligomerization mediated by the RBCC/TRIM motif is essential for the transformation potential of the PML-RARα fusion protein.

The Nuclear Dot Protein Sp100, Characterization of Domains Necessary for Dimerization, Subcellular Localization, and Modification by Small Ubiquitin-like Modifiers

The Sp100 and promyelocytic leukemia proteins (PML) are constituents of nuclear domains, known as nuclear dots (NDs) or PML bodies, and are both covalently modified by the small ubiquitin-related protein SUMO-1. NDs play a role in autoimmunity, virus infections, and in the etiology of acute promyelocytic leukemia. To date, little is known about the function of the Sp100 protein. Here we analyzed Sp100 domains that determine its subcellular localization, dimerization, and SUMOylation. A functional nuclear localization signal and an ND-targeting region that coincides with an Sp100 homodimerization domain were mapped. Sequences similar to the Sp100 homodimerization/ND-targeting region occur in several other proteins and constitute a novel protein motif, termed HSR domain. The lysine residue of the Sp100 protein, to which SUMO-1 is covalently linked, was mapped within and may therefore modulate the previously described HP1 protein-binding site. A consensus sequence for SUMOylation of proteins in general is suggested. SUMOylation strictly depended on a functional nuclear localization signal but was not necessary for nuclear import or ND targeting. A three-dimensional structure of Sp100, which supports the mapping data and provides additional information on Sp100 structure/function relationships, was generated by computer modeling. Taken together, our studies indicate the existence of well defined Sp100 domains with functions in ND targeting, nuclear import, nuclear SUMOylation, and protein-protein interaction.

Nuclear dots: actors on many stages.

Nuclear dots (NDs), alternatively designated nuclear bodies (NBs), PML oncogenic domains (PODs), nuclear domain 10 (ND10) or Kr-bodies, became a major topic for researchers in many fields only recently. Originally described as an autoantigenic target in patients with primary biliary cirrhosis, they are now also known to play a role in development of acute promyelocytic leukemia (APL) and possibly other forms of neoplasia. Size, number and composition of NDs are regulated throughout the cell cycle. Infection with herpes simplex virus, adenovirus, cytomegalovirus, Epstein-Barr-virus, influenza virus and human T cell lymphotropic virus type I (HTLV I) strongly modifies ND structure through viral regulatory proteins. Due to this finding and because at least three of the cellular ND proteins are highly interferon-inducible, a function of NDs in early viral infection or in antiviral response has been postulated. Functional data are currently available only for two of the ND-associated proteins. The Sp100 protein seems to have transcriptional transactivating property, whereas the promyelocytic leukemia protein (PML) was reported to suppress growth and transformation. Here, we give a brief overview of the data currently available on NDs. Thus, we hope to link seemingly unrelated findings in the literature on oncology, virology, cell biology and immunology.

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology.

FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies

Caspase‐8‐binding protein FLICE‐associated huge protein (FLASH) has been proposed to regulate death receptor CD95‐induced apoptosis through facilitating caspase‐8 activation at the death‐inducing signaling complex. Here, we found that FLASH interacts with the PML nuclear body component Sp100 and predominantly resides in the nucleus and nuclear bodies (NBs). In response to CD95 activation, FLASH leaves the NBs and translocates into the cytoplasm where it accumulates at mitochondria. The nucleo‐cytoplasmic translocation of FLASH requires CD95‐induced caspase activation and is facilitated by the Crm1‐dependent nuclear export pathway. Downregulation of FLASH by RNA interference or inhibition of its nucleo‐cytoplasmic shuttling reduced CD95‐induced apoptosis. Furthermore, we show that the adenoviral anti‐apoptotic Bcl‐2 family member E1B19K traps FLASH and procaspase‐8 in a ternary complex at mitochondria, thereby blocking CD95‐induced caspase‐8 activation. Knock‐down of Sp100 potentiated CD95‐activated apoptosis through enhancing nucleo‐cytoplasmic FLASH translocation. In summary, our findings suggest that CD95 signals via a previously unrecognized nuclear pathway mediated by nucleo‐cytoplasmic translocation of FLASH.

The Interferon (IFN)-stimulated Gene Sp100 Promoter Contains an IFN-γ Activation Site and an Imperfect IFN-stimulated Response Element Which Mediate Type I IFN Inducibility

Expression of the nuclear domain-associated proteins Sp100, PML, and NDP52, is enhanced by interferons (IFNs) on the mRNA and protein level. Increase both of Sp100 and PML mRNA is due to enhanced transcription of the corresponding genes which occurs independently of cellular protein synthesis immediately upon IFN-β addition. Here, we describe the molecular cloning and functional analysis of the Sp100 promoter. DNA sequence analysis revealed potential binding sites for several constitutive and IFN-inducible transcription factors. Consistent with the absence of a TATA box and an initiator element, several transcription initiation sites were found. Transient expression studies identified an imperfect IFN-stimulated response element within the first 100 nucleotides upstream of the major transcription start site. This element rendered a heterologous promoter IFN-β-inducible and bound IFN-stimulated gene factor 2 strongly but IFN-stimulated gene factor 3 only weakly. An IFN-γ activation site approximately 500 base pairs upstream of the IFN-stimulated response element was found to bind three IFN-α/β activation factors upon IFN-β induction and conferred both type I and type II IFN inducibility upon a heterologous promoter. These data demonstrate a novel arrangement of a nonoverlapping IFN-γ activation site and an IFN-stimulated response element mediating type I IFN inducibility, previously not reported for other IFN-stimulable promoters.

Ap Endonuclease Paralogues with Distinct Activities in DNA Repair and Bacterial Pathogenesis.

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3′‐phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose‐binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.

Segmentation of Fluorescence Microscopy Images for Quantitative Analysis of Cell Nuclear Architecture

Considerable advances in microscopy, biophysics, and cell biology have provided a wealth of imaging data describing the functional organization of the cell nucleus. Until recently, cell nuclear architecture has largely been assessed by subjective visual inspection of fluorescently labeled components imaged by the optical microscope. This approach is inadequate to fully quantify spatial associations, especially when the patterns are indistinct, irregular, or highly punctate. Accurate image processing techniques as well as statistical and computational tools are thus necessary to interpret this data if meaningful spatial-function relationships are to be established. Here, we have developed a thresholding algorithm, stable count thresholding (SCT), to segment nuclear compartments in confocal laser scanning microscopy image stacks to facilitate objective and quantitative analysis of the three-dimensional organization of these objects using formal statistical methods. We validate the efficacy and performance of the SCT algorithm using real images of immunofluorescently stained nuclear compartments and fluorescent beads as well as simulated images. In all three cases, the SCT algorithm delivers a segmentation that is far better than standard thresholding methods, and more importantly, is comparable to manual thresholding results. By applying the SCT algorithm and statistical analysis, we quantify the spatial configuration of promyelocytic leukemia nuclear bodies with respect to irregular-shaped SC35 domains. We show that the compartments are closer than expected under a null model for their spatial point distribution, and furthermore that their spatial association varies according to cell state. The methods reported are general and can readily be applied to quantify the spatial interactions of other nuclear compartments.

Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

Significance Bacterial cellulose is a remarkable material that is malleable, biocompatible, and over 10-times stronger than plant-based cellulose. It is currently used to create materials for tissue engineering, medicine, defense, electronics, acoustics, and fabrics. We describe here a bacterial strain that is readily amenable to genetic engineering and produces high quantities of bacterial cellulose in low-cost media. To reprogram this organism for biotechnology applications, we created a set of genetic tools that enables biosynthesis of patterned cellulose, functionalization of the cellulose surface with proteins, and tunable control over cellulose production. This greatly expands our ability to control and engineer new cellulose-based biomaterials, offering numerous applications for basic research, materials science, and biotechnology. Bacterial cellulose is a strong and ultrapure form of cellulose produced naturally by several species of the Acetobacteraceae. Its high strength, purity, and biocompatibility make it of great interest to materials science; however, precise control of its biosynthesis has remained a challenge for biotechnology. Here we isolate a strain of Komagataeibacter rhaeticus (K. rhaeticus iGEM) that can produce cellulose at high yields, grow in low-nitrogen conditions, and is highly resistant to toxic chemicals. We achieved external control over its bacterial cellulose production through development of a modular genetic toolkit that enables rational reprogramming of the cell. To further its use as an organism for biotechnology, we sequenced its genome and demonstrate genetic circuits that enable functionalization and patterning of heterologous gene expression within the cellulose matrix. This work lays the foundations for using genetic engineering to produce cellulose-based materials, with numerous applications in basic science, materials engineering, and biotechnology.

A highly amplified mouse gene is homologous to the human interferon-responsive Sp100 gene encoding an autoantigen associated with nuclear dots

In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment.