Visualization, analysis, and design of COMBO-FISH probes in the grid-based GLOBE 3D genome platform

Biophysical Genomics, Dept. Cell Biology & Genetics, Erasmus MC, Dr. Molewaterplein, Rotterdam, The Netherlands.
Studies in health technology and informatics 07/2010; 159:171-80. DOI: 10.3233/978-1-60750-583-9-171
Source: PubMed


The genome architecture in cell nuclei plays an important role in modern microscopy for the monitoring of medical diagnosis and therapy since changes of function and dynamics of genes are interlinked with changing geometrical parameters. The planning of corresponding diagnostic experiments and their imaging is a complex and often interactive IT intensive challenge and thus makes high-performance grids a necessity. To detect genetic changes we recently developed a new form of fluorescence in situ hybridization (FISH) - COMBinatorial Oligonucleotide FISH (COMBO-FISH) - which labels small nucleotide sequences clustering at a desired genomic location. To achieve a unique hybridization spot other side clusters have to be excluded. Therefore, we have designed an interactive pipeline using the grid-based GLOBE 3D Genome Viewer and Platform to design and display different labelling variants of candidate probe sets. Thus, we have created a grid-based virtual "paper" tool for easy interactive calculation, analysis, management, and representation for COMBO-FISH probe design with many an advantage: Since all the calculations and analysis run in a grid, one can instantly and with great visual ease locate duplications of gene subsequences to guide the elimination of side clustering sequences during the probe design process, as well as get at least an impression of the 3D architectural embedding of the respective chromosome region, which is of major importance to estimate the hybridization probe dynamics. Beyond, even several people at different locations could work on the same process in a team wise manner. Consequently, we present how a complex interactive process can profit from grid infrastructure technology using our unique GLOBE 3D Genome Platform gateway towards a real interactive curative diagnosis planning and therapy monitoring.

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    • "Probe sets for double-helical or for triple-helical hybridization can be designed [8]. In order to obtain a specific label of a given chromatin target with short oligonucleotides, it is necessary to first identify candidate target sites and second to test these for reoccurrences against the complete human genome database by means of bioinformatic investigations [7,8,10,11]. By this means only those target sites are selected for a given gene locus that specifically colocalize at this region of interest, i.e., the individual target sites may occur at several loci in the whole genome; however, all selected target sites only occur conjointly at the given genome locus. "
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    ABSTRACT: With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15-30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)-synthetic DNA analogues with a neutral backbone-which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488(®) showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10-20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
    International Journal of Molecular Sciences 10/2010; 11(10):4094-105. DOI:10.3390/ijms11104094 · 2.86 Impact Factor
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    ABSTRACT: Long Range Chromatin Interactions within the m-Globin Locus Lately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors (TF) to TF binding sites also becomes an important factor to the gene's activation status. Both such changes have been ascribed to the mouse -haemoglobin gene cluster (Fig. 1A) as a trigger to activate globin expression in the erythroid cell lineage. Early models speculated a scanning, random activation or a looping mechanism to activate globin transcription. The chromatin conformation capture (3C) technique has shown that there is a molecular interaction between various DNAse I hypersensitive (HS) sites that are located up-and downstream of the -globin gene cluster, the HS sites of the Locus Control Region (LCR) and the promoter by means of a dynamic looping mechanism. The clustering of the HS sites of the LCR and the up-and downstream HS sites results in the formation of a so called Active Chromatin Hub (ACH) which is depending on at least two erythoid TF: EKLF and GATA-1 (Fig. 1B-C). The long range interactions between the outlying HS-84/-85,-62/-60 and the 3'HS1 are depending on the presence of CTCF, a TF that is thought to play an important role in long range chromatin interactions across the whole genome. Prior to gene activation, cells of the early erythroid lineage (progenitors) already show a presence of an ACH (Fig. 1B), which is not found in non-erythroid cells. The final chromatin 3D structure consist of four major loops sizing 25-38Kb and two minor loops within the LCR sizing 4.5 and 12Kb (Fig. 1A, D). To confirm this looping hypothesis (based on 3C technology) we used an in situ hybridization approach to visualize and, after image restoration, quantitatively measure the 3D conformational changes that take place within the locus in erythroid cells before and after differentiation. 1A DNAse I hypersensitive sites masked DNA sequence Visualizing Long Range Chromatin Interactions 3D-DNA fluorescence in situ hybridization (FISH) method was used to visualize chromatin structure of the m-globin locus in undifferentiated (inactive chromatin) and differentiated (active chromatin) mouse erythroleukemia (MEL) cells. Thereto we designed PCR probes that cover unique DNA sequences of 128Kb or 175Kb region (Fig. 1A). The 3D conformation of the 128Kb (Fig. 2B, left) and 175Kb (Fig. 2B, right) probed DNA region according to the 3C technique is shown in Figure 2. 3D high resolution images were acquired with a Leica SP5 confocal microscope. For quantitative measurements, all images were acquired with identical configuration and settings. 100nm beads were imaged to determine the point spread function (PSF). Images were restored by deconvolution using the classic maximum of likelihood algorithm (Huygens Professional, v3.5) and an empirically obtained PSF. Images were volume rendered in 3D using the object tool analyzer (Fig. 5) and geometric sizes (lateral and axial length, volume, surface) and shape (axial sphericity, object angularity) of each m-globin locus were determined.
    EMBO Workshop on Visualizing Biological Data (VIZBIO), EMBL Heidelberg, Heidelberg, Germany, 3rd - 5th March, 2010.; 03/2010
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    ABSTRACT: Here we show a 3D DNA-FISH method to visualizes the 3D structure of the β-globin locus. Geometric size and shape measurements of the 3D rendered signals (128Kb) show that the volume of the β-globin locus decreases almost two fold upon gene activation. A decrease in length and a distinctive change in shape and surface structure of the locus are also observed. Adding 5’ and 3’end regions to the probe (175Kb) showed a less prominent change in length, shape and structure. It was shown (data not on this poster) that the physical distance between the two flanking regions shift in a similar limited manner, indicating that the flanking regions do not participate in ACH formation and thus active chromatin folding is occurring only within the locus proper.
    10th international ELMI Meeting, EMBO Workshop on Advanced Light Microscopy Techniques and their Applications, EMBL Heidelberg, Heidelberg, Germany, 18th - 21th June, 2010.; 06/2010
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