In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G 1 and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells.chromatin ͉ DNA replication ͉ EBNA1 ͉ Epstein-Barr virus ͉ Orc6

Eukaryotic cells devoid of mitochondrial DNA (ρ0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. ρ0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of ρ0 cell lines, we developed an extremely mild, reliable and timesaving method to generate ρ0 cell lines within 3–5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK− the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat ρ0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK− ρ0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human ρ0 mitochondria.

Human mesenchymal stromal cells derived from bone marrow (BMSC) and adipose tissue (ATSC) represent a valuable source of progenitor cells for cell therapy and tissue engineering. While ectopic bone formation is a standard activity of human BMSC on calcium phosphate ceramics, the bone formation capacity of human ATSC has so far been unclear. The objectives of this study were to assess the therapeutic potency of ATSC for bone formation in an ectopic mouse model and determine molecular differences by standardized comparison with BMSC. Although ATSC contained less CD146(+) cells, exhibited better proliferation and displayed similar alkaline phosphatase activity upon osteogenic in vitro differentiation, cells did not develop into bone-depositing osteoblasts on β-TCP after 8weeks in vivo. Additionally, ATSC expressed less BMP-2, BMP-4, VEGF, angiopoietin and IL-6 and more adiponectin mRNA, altogether suggesting insufficient osteochondral commitment and reduced proangiogenic activity. Chondrogenic pre-induction of ATSC/β-TCP constructs with TGF-β and BMP-6 initiated ectopic bone formation in >75% of samples. Both chondrogenic pre-induction and the osteoconductive microenvironment of β-TCP were necessary for ectopic bone formation by ATSC pointing towards a need for inductive conditions/biomaterials to make this more easily accessible cell source attractive for future applications in bone regeneration.

Retinitis pigmentosa (RP) is a common hereditary eye disease that causes blindness due to a progressive loss of photoreceptors in the retina. RP can be elicited by mutations that affect the tri-snRNP subunit of the pre-mRNA splicing machinery, but how defects in this essential macromolecular complex transform into a photoreceptor-specific phenotype is unknown. We have modeled the disease in zebrafish by silencing the RP-associated splicing factor Prpf31 and observed detrimental effects on visual function and photoreceptor morphology. Despite reducing the level of a constitutive splicing factor, no general defects in gene expression were found. Instead, retinal genes were selectively affected, providing the first in vivo link between mutations in splicing factors and the RP phenotype. Silencing of Prpf4, a splicing factor hitherto unrelated to RP, evoked the same defects in vision, photoreceptor morphology and retinal gene expression. Hence, various routes affecting the tri-snRNP can elicit tissue-specific gene expression defects and lead to the RP phenotype.

Extensive epidemiological data indicate that inorganic arsenic is associated with several types of human cancer. Nevertheless, the underlying mechanisms are poorly understood. Among its mode of action are the alterations on DNA methylation, which provoke aberrant gene expression. However, beyond DNA methylation, little is known about arsenic's effects on chromatin. In this study, we investigated the effects of sodium arsenite (NaAsO(2)) on global histone modifications and nucleosome-associated proteins. Our findings revealed that NaAsO(2) exposure significantly increases global histone acetylation. This effect was related to the inhibition of histone deacetylase (HDAC) activity because NaAsO(2) was able to inhibit HDACs comparable to the well-known HDAC inhibitor trichostatin A (TSA). Furthermore, analyses of the dynamic properties of the nucleosome-associated high mobility group N proteins demonstrate that NaAsO(2) elevates their mobility. Thus, our data suggest that NaAsO(2) induces chromatin opening by histone hyperacetylation due to HDAC inhibition and increase of the mobility of nucleosome-associated proteins. As the chromatin compaction is crucial for the regulation of gene expression as well as for genome stability, we propose that chromatin opening by NaAsO(2) may play a significant role to impart its genotoxic effects.

BackgroundHigh mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation.ResultsWe found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable over-expression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells.ConclusionsOur data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.

BackgroundRetinitis pigmentosa (RP) is an inherited eye disease characterized by the progressive degeneration of rod photoreceptor cells. Mutations in pre-mRNA splicing factors including PRPF31 have been identified as cause for RP, raising the question how mutations in general factors lead to tissue specific defects.ResultsWe have recently shown that the zebrafish serves as an excellent model allowing the recapitulation of key events of RP. Here we use this model to investigate two pathogenic mutations in PRPF31, SP117 and AD5, causing the autosomal dominant form of RP. We show that SP117 leads to an unstable protein that is mislocalized to the rod cytoplasm. Importantly, its overexpression does not result in photoreceptor degeneration suggesting haploinsufficiency as the underlying cause in human RP patients carrying SP117. In contrast, overexpression of AD5 results in embryonic lethality, which can be rescued by wild-type Prpf31. Transgenic retina-specific expression of AD5 reveals that stable AD5 protein is initially localized in the nucleus but later found in the cytoplasm concurrent with progressing rod outer segment degeneration and apoptosis. Importantly, we show for the first time in vivo that retinal transcripts are wrongly spliced in adult transgenic retinas expressing AD5 and exhibiting increased apoptosis in rod photoreceptors.ConclusionOur data suggest that distinct mutations in Prpf31 can lead to photoreceptor degeneration through different mechanisms, by haploinsufficiency or dominant-negative effects. Analyzing the AD5 effects in our animal model in vivo, our data imply that aberrant splicing of distinct retinal transcripts contributes to the observed retina defects.

PURPOSE. In experimental eye research, zebrafish has become a powerful model for human retina disorders. The purpose of the present study is the characterization of antibodies commonly employed in zebrafish models for rod photoreceptor degeneration.METHODS. The 1D4 monoclonal antibody, developed against bovine rhodopsin, has been widely used in studies addressing structural and functional features of rhodopsin and was reported as an informative marker to stain rod outer segments in both mice and zebrafish. We have used transgenic reporter lines and histologic analysis to determine the photoreceptor types identified by 1D4 and other antibodies in zebrafish.RESULTS. We demonstrate that 1D4, in contrast to what has been reported previously, does not recognize rod outer segments in zebrafish, but instead labels long double cone outer segments consistent with sequence conservation of the respective epitope. As an alternative marker for zebrafish rods, we characterized the monoclonal antibody zpr-3, which was found to stain outer segments of both rods, as well as double cones.CONCLUSIONS. Our findings highlight the importance to confirm specificity of antibodies in cross-species experiments for correct interpretation of experimental data. Our findings clarify conflicting published information arising from studies using 1D4 and zpr-3 antibodies in zebrafish. (Invest Ophthalmol Vis Sci. 2012;53:4943-4951)