164 research outputs found
Nuclear factor I genomic binding associates with chromatin boundaries
No full textBACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation. NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871
First record of the Red Sea immigrant Chrysallida fischeri (Hornung and Mermod, 1925) (Gastropoda: Pyramidellidae) from Greek waters
No full textThe Red Sea mollusc Chrysallida fischeri (Hornung and Mermod, 1925) was collected in 2005 off Makri, Rhodes, Greece. This is the first record of the species in the Aegean Sea. The presence of both live specimens and shells suggest that C. fischeri is well established in the area. © 2007 The Author(s)
Study of DNA repair and recombination mechanisms in Chinese hamster ovary cells
No full textThe CRISPR nuclease systems greatly facilitate targeted genome modifications in mammalian cells. The outcome of genome editing depends on the involved DNA double strand break (DSB) repair pathways. While the classical non-homologous end-joining and the poorly defined alternative end-joining (alt-EJ) DSB repair pathways can cause imprecise repair and thus gene inactivations, the homologous recombination (HR) pathway often introduces precise modifications. Although CRISPR is highly efficient at inactivating single genes, it is inefficient at introducing precise genome modifications. Moreover, its efficiency at inactivating multi-locus DNA sequences such as highly repetitive endogenous viral elements also remains limited.
This thesis addressed these limitations by better characterizing DSB repair pathways in Chinese hamster ovary (CHO) cells - the most widely used production cell host for therapeutic proteins. In this thesis, I first aimed at identifying rate-limiting factors to improve HR-mediated genome editing. Second, I strove for studying approaches to inactivate repetitive endogenous retroviruses (ERV) presumably releasing viral particles into the CHO supernatant.
To identify factors limiting HR, we established two chromosomal CHO assays that measure HR activity based on the correction of a GFP loss-of-function mutation. By using knockdown and overexpression studies, we found that efficient HR-mediated genome editing depended on certain alt-EJ activities. Furthermore, we observed that alt-EJ contribution to HR correlates with the nuclease type and the location of the DSB site relative to the GFP mutation. These observations suggest that alt-EJ and HR repair pathways tightly interact and challenges the common perception of alt-EJ opposing HR. Finally, among the tested repair factors, high Mre11 nuclease and Pari anti-recombinase as well as low Rad51 recombinase levels were the most rate-limiting factors for HR in CHO cells. Counteracting these bottlenecks improved HR efficiency by 75%.
To inactivate repetitive ERVs, we transiently expressed a CRISPR-Cas9 nuclease that targets the gag gene of a specific transcriptionally active ERV group. Clones bearing a loss-of-function mutation in one particular ERV locus and corresponding mRNA produced considerably fewer particles loaded with viral RNA genomes. These findings indicated that a single ERV locus is responsible for the release of most, if not all, viral particles from CHO cells. Notably, ERV mutagenesis did not compromise cell growth, cell size or therapeutic protein production. In sum, this work provided novel strategies to improve HR-mediated genome editing and to inhibit viral particle release from CHO cells
Statistical and experimental analysis of quantitative PCR
No full textQuantitative PCR (qPCR) is a powerful technique that is now commonly used in many research and clinical laboratories. Although it allows precise quantification of specific DNA sequences, it is often not used at its full potential. A number of data collection and processing strategies have been described for the implementation of quantitative PCR. However, they can be experimentally cumbersome, their relative performances have not been evaluated systematically, and they often remain poorly validated statistically and/or experimentally. Highly sophisticated mathematical models have also been proposed to decipher the PCR intimate process, but most of them were never validated. Often poor knowledge of the underlying mechanisms lead to inaccurate results and misinterpretation. Our first objective has been to measure by qPCR chromatin accessibility that was probed by a DNA adenine methylase. Results showed that 2-fold variations in relative accessibility could be assessed. However, the variability of the measures lead us to question the reproducibility of the qPCR. Placing our work in a robust statistical frame work, we proceeded with the evaluation of the parameters influencing PCR efficiency. Performance of known methods were also evaluated in terms of sensitivity, precision and robustness; and compared with various improved models, based on individual or averaged efficiency values. Our results show that when accurate quantification is required, single reaction efficiencies need to be measured and averaged for a given sample and primer. Furthermore, we show that well designed primers can hold the assumption of equal efficiency and therefore that the ∆Ct model is valid for measurement of 5-fold induction of a gene expression, at the least. Finally, we tried, as much as we could, to produce an exhaustive list of pitfalls qPCR users could stumble upon and proposed solutions. Our results allow the precise evaluation of minute amount of DNA, with a predictable and realistic number of measures
Assessment of a cell therapy approach for Duchenne muscular dystrophy using mesoangioblasts and non-viral vectors
No full textAnalyse des modifications de la chromatine impliquées dans l'effet barrière de CTF1 aux télomères humains
No full textEukaryotic genomes are compartmentalized in different structural domains that can affect positively or negatively gene expression. These regions of euchromatin and heterochromatin are characterized by distinct histones marks which can facilitate or repress gene transcription. The chromatin environment represents thus one of the main problems to control gene expression in biotechnological applications or gene therapy, since its expression is affected by the chromatin neighboring its locus of insertion. Some chromatin regions like telomeres are composed of constitutive heterochromatin which leads to the telomeric position effect (TPE) that silences genes adjacent to the telomere. TPE is known to spread by the selfrecruitment of the SIR histone deacetylase complex from the telomere in S.cerevisiae, but the histone marks that are associated to telomeric chromatin in mammalian cells remain mostly unknown. The transcription factor CTF1 has shown antisilencing properties in mammalian cells and also a boundary activity against TPE in yeast cells when fused to the yeast Gal4 DNA binding domain. In the work presented here, we describe a dual-reporter system to assess the boundary activity of proteins such as CTF1 at human telomeres. When located between the two reporter genes, CTF1 shields the telomere distal gene from TPE, while the telomereproximal gene remains silenced by telomeric heterochromatin. The boundary activity of CTF1 is shown to act regardless its function of transcriptional activator, by opposition to the transcriptional activator VP16 which activates indifferently both transgenes. Moreover, this study shows that CTF1 boundary activity is linked to its H3 binding function, as expected from a chromatin remodeler. ChIP experiments showed that histone deacetylation is the main histone modification involved in gene silencing at mammalian cell telomeres. Distinctly to yeast cells, the histone deacetylation signal in human cells extented over a short range along the chromosome. CTF1 may help to block this propagation and therefore to restore histones acetylation level on telomere protected locus. Surprisingly, other histone marks such as trimethyl-H3K9 or trimethyl-H4K20 were found on telomere protected locus, while in another clone, unsilencing of telomere distal transgene was associated with recruitment of the histone variant H2A.Z. Thus, I conclude that CTF1 displays a chromatin boundary function which is independent of its transcriptional activity and therefore exhibit features required for use as chromatin insulator in biotechnological applications.
RESUME
Les génomes eucaryotes sont compartementalisés en domaines structurels qui peuvent
affecter positivement ou négativement l'expression des gènes avoisinants. Ces régions dites
d'euchromatine ou d'hétérochromatine sont caractérisées par des modifications posttraductionnelles
des histones qui peuvent faciliter ou au contraire inhiber la transcription des
gènes qui s'y trouvent. Ainsi, isoler un gène de son environnement chromatinien est problème
fréquent lorsqu'il s'agit de contrôler son expression dans le cadre d'applications en
biotechnologie ou encore en thérapie génique. Certaines régions de chromatine telles que les
télomères sont composées d'hétérochromatine constitutive qui mène au silençage des gènes
avoisinants. Cet effet de position télomérique (TPE) est connu dans la levure S.cerevisiae
comme se propageant par auto-recrutement du complexe de déacétylation d'histone SIR, alors
que peu de modifications de chromatine ont pu être associées à ce phénomène dans les
cellules de mammifères.
Le facteur de transcription CTF1 a montré des propriétés d'anti-silençage dans les
cellules de mammifères, ainsi qu'une activité barrière contre le silençage télomérique dans les
cellules de levures lorsqu'il est fusionné au domaine de liaison à l'ADN de la protéine de
levure Gal4. Dans le travail présenté ci-après est décrit un système à deux gènes rapporteurs
permettant de mesurer l'activité barrière de protéines telles que CTF1 aux télomères humains,
et les modifications de chromatine qui y sont associées. Lorsque CTF1 est placé entre les
deux gènes rapporteurs, le gène distant du télomère est protégé du silençage qui lui est
associé, alors que le gène proche du télomère reste soumis à ce silençage induit par
l'hétérochromatine télomérique. L'activité barrière de CTF1 est montrée ici comme agissant
indépendamment de son activité transcriptionnelle, par opposition à l'activateur
transcriptionnel VP16 qui active indifféremment les deux transgènes. En outre, cette étude
appuie l'hypothèse stipulant que CTF1 agisse comme remodeleur chromatinien puisqu'elle
démontre que son activité barrière est directement dépendante de son activité de liaison avec
l'histone H3.
De plus, des expériences d'immuno-précipitation de la chromatine démontrent que la
déacétylation des histones est le majeur phénomène intervenant dans le silençage télomérique.
Par opposition à la levure, ce signal de déacétylation ne se propage dans les cellules humaines
que sur une courte distance le long du chromosome. CTF1 agit ainsi en bloquant cette
propagation et en restaurant le niveau d'acétylation des histones sur le locus protégé du
télomère. De manière surprenante et inattendue, d'autres modifications d'histones telles que
4
les H3K9 et H4K20 triméthylées sont aussi observées à ce locus, tandis le recrutement du
variant H2A.Z peut aussi être suffisant à restaurer l'expression du gène distant du télomère.
En terme de cette analyse, CTF1 exhibe ainsi une fonction de barrière chromatinienne
qui exclue une activité transcriptionnelle non désirée - propriété qui est requise dans
l'établissement des isolateurs visant à permettre le contrôle d'un transgène dans le cadre
d'applications en biotechnologies
Identification of genetic insulator elements to increase the safety of viral gene therapy vectors
No full textHaematopoeitic system diseases, both acquired and inherited, can be currently cured by allogeneic haematopoietic stem cell transplantation. This treatment provides highly successful immune function recovery for patients receiving grafts of HLA-compatible donors but has still a great risk of complications and even failure if no suitable donor is available. Among the alternative therapeutic options, ex vivo retrovirus-mediated gene transfer into haematopoietic progenitor cells has been shown to be an efficient strategy for a substantial number of severe combined immunodeficiency-suffering patients. A recent gene therapy trial has been remarkably effective for the immunological reconstitution of patients suffering from X-linked severe combined immunodeficiency. This treatment was able to provide full correction of disease phenotype and thus, clinical benefit. However, the appearance of leukemia in several patients has put in question the safety of the procedure. This severe adverse event has been attributed to the integration of the therapeutic transgene-carrying viral vector into a known T-cell oncogene, LMO2, thereby contributing to the development of T-cell leukemia by causing aberrant expression of LMO2. Further studies mentioned the possible retroviral-mediated cis-activation of the LMO2 promoter underlying the potential ability of retroviral regulatory elements to influence neighboring gene transcription. This project aimed at decreasing the risk associated with the use of viral vectors for gene therapy through the identification of genetic insulator elements capable of isolating the vector regulatory elements in order to prevent the activation of chromosomal genes by the viral enhancers. We have established a standardized screening procedure whereby the potency of insulators can be assessed quantitatively on relevant vector elements. This assay system consists of a series of plasmids containing two reporter genes: one mimicking a therapeutic gene under the control of strong viral long terminal repeat (LTR) enhancer, and the other one standing for an endogenous gene close to the chromosomal vector integration site. The assay allowed the quantification of the enhancer blocking activity of the well-characterized chicken beta-globin 5'HS4 insulator (cHS4) in cultured cells. We assessed the insulator activity of novel synthetic elements, constructed from optimized binding sites for the insulator protein CTCF. In addition, we demonstrated the enhancer blocking activity of the nuclear factor 1 protein family (CTF/NF1) and showed that it also displays barrier properties, protecting transgene expression from silencing. We showed that both CTCF and CTF/NF1 binding sites act as insulators that mediate potent enhancer-blocker activity, resulting in a commensurate reduction of genotoxicity when implemented in viral gene therapy vectors. Finally, we used the same approach to analyze the enhancer blocking activity of well-known chromatin domain delimiters, matrix attachment regions (MAR), mainly characterized by their barrier properties. We could demonstrate the enhancer blocking ability of the 1-68 MAR that is mainly harbored by an A-T rich core sub-region.LMR
Protein-binding microarray technology and ChIP-sequencing for the identification of novel targets of the human tumor suppressor AP2[alpha]
No full textThe mis-regulation of gene transcription in the living cell can be the cause of complex human diseases like cancer. One challenge of the post-genomic era of biomedical research is the development of in vitro techniques for the rapid and cost-effective analysis of regulatory transcriptional patterns of disease. Two crucial points for this endeavour have to be considered: (1) Sensitivity, since biologically active transcription factors are expressed at low amounts in the nucleus of the living cell and (2) the as close as feasible reconstitution of the nuclear microenvironment in order to guarantee as close to natural conditions for the performance of experiments as possible. The first part of this thesis reports on the application of protein-binding microarrays (PBM) bearing long-stranded DNA molecules for the analysis of a human disease-related protein, the human tumor suppressor activating enhancer-binding protein 2 alpha (AP2α). The second part of this thesis describes how chromatin-immunoprecipitation combined with high-throughput sequencing (ChIP-seq) and subsequent data analysis led to the discovery of a novel AP2α-binding motif in a cancer cell line model. We first developed a setup and protocol to perform on-chip protein-DNA molecule interaction analyses. Following this, recombinant AP2α protein was produced and purified using an E.coli host system. AP2α was subsequently assessed for its binding specificity to an array of ∼6000 human promoter and intergenic sequences. These experiments confirmed previously established AP2α DNA binding sequences and allowed the identification of novel ones. High and low binder sequences were selected from the PBM data and analyzed for specific AP2α binding in vivo utilizing reporter transfection and chromatin-immunoprecipitation (ChIP) assays. In contrast to the lowest AP2α-bound sequences, the highest PBM-bound sequences had significant effects on reporter gene expression. ChIP-qPCR revealed these sequences were bound by AP2α in vivo. From these results Kallikrein 5, a member of the kallikrein family of extracellular proteases that includes the prostate-specific antigen (PSA), which is currently emerging as one of the most prominent biomarkers of tumor progression for various types of cancers, and the growth-arrest specific 2 (GAS2) protein, which modulates cell susceptibility to p53-dependent apoptosis, were identified as novel AP2α targets. Finally, the binding patterns of AP2α from nuclear extracts of breast cancer tissue biopsies were assessed on the 6k human promoter arrays and compared to the binding patterns of 'healthy' tissue extracts. Overall these experiments confirmed known targets of AP2α. Interestingly a discrepancy of 26% was revealed when assessing the correlation of binding data from healthy and tumor tissue biopsies. This finding suggested that depending on the source of the protein, that is of either healthy or tumor tissue origin, the DNA-binding specificity of the protein factor under study might be altered and most importantly this change in binding specificity can be monitored by PBM. The second part of this thesis describes the analysis of the binding specificity of AP2α by combining chromatin-immunoprecipitation with high-throughput sequencing (ChIP-seq). The binding specificity of the human tumor suppressor protein AP2α in the living cell was assessed in a colon cancer cell model. Analysis of the ChIP-seq enriched sequences resulted in the discovery of a novel recognition motif for the in vivo binding of AP2α. Interestingly, this sequence motif resembles the motifs discovered from data sets with recombinant protein alone. Further analysis of a previously described in vitro and the new ChIP-seq discovered binding motifs were performed by electrophoretic mobility shift assays, which demonstrated that the newly discovered motif shows the same effects with colon cancer and breast cancer nuclear extracts, thereby suggesting a general relevance of the motif independent of the cell type. In these DNA-binding competition experiments (EMSA), the previously computed, in vitro derived DNA recognition sequences does not abolish native AP2α protein (obtained from crude nuclear cell extracts) interaction with the ChIP-seq discovered motif. This observation indicates for a complex situation in vivo that might require protein co-factors in order to allow specific AP2α-DNA interaction. Therefore additional experiments will be required in order to assess the novel motif further. In conclusion, this thesis work demonstrates that protein-binding microarrays (PBM) can be used for the analysis of binding patterns of a human disease-relevant protein. It could be shown that the PBM setup developed was capable of identifying novel cancer-linked target genes for AP2α. It was also shown to be sensitive enough to directly probe nuclear extracts from tissue biopsies for specific DNA-binding. Finally, a novel DNA-recognition motif for AP2α was discovered from cellular assays. Additional experiments are required to assess whether this motif can be utilized for the reliable assignment of novel probes that may subsequently be integrated onto protein-binding microarrays. Together with the analyses for additional protein markers of disease, this avenue might serve as a platform for developing PBM technology further towards biotechnological applications such as diagnostics of regulatory transcriptional markers and patterns of cancer.LMR
Gene-mediated restoration of normal myofiber elasticity in dystrophic muscles
No full textDystrophin mediates a physical link between the cytoskeleton of muscle fibers and the extracellular matrix, and its absence leads to muscle degeneration and dystrophy. In this article, we show that the lack of dystrophin affects the elasticity of individual fibers within muscle tissue explants, as probed using atomic force microscopy (AFM), providing a sensitive and quantitative description of the properties of normal and dystrophic myofibers. The rescue of dystrophin expression by exon skipping or by the ectopic expression of the utrophin analogue normalized the elasticity of dystrophic muscles, and these effects were commensurate to the functional recovery of whole muscle strength. However, a more homogeneous and widespread restoration of normal elasticity was obtained by the exon-skipping approach when comparing individual myofibers. AFM may thus provide a quantification of the functional benefit of gene therapies from live tissues coupled to single-cell resolution
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