The GLOBE-Consortium: The Next-Generation Genome Viewer

The GLOBE 3D Genome Viewer is the novel system-biology oriented genome browser necessary to access, present, annotate, and to simulate the holistic genome complexity in a unique gateway towards a real understanding, educative presentation and curative manipulation planning of this tremendous evolutionary information grail – genomes. This has required completely new approaches to represent the genome architecture realistically in combination with the various types of informational annotation including experimental data or instant analysis capabilities. This creates unrivalled new opportunities for scientific researchers, diagnostic users, educators and publishers as well as PR and commercial applicants. Potential BETA-TESTERS of the GLOBE 3D Genome Viewer are asked to sign up now

The system-biological GLOBE 3D Genome Platform.

Genomes are tremendous co-evolutionary holistic systems for molecular storage, processing and fabrication of information. Their system-biological complexity remains, however, still largely mysterious, despite immense sequencing achievements and huge advances in the understanding of the general sequential, three-dimensional and regulatory organization. Here, we present the GLOBE 3D Genome Platform a completely novel grid based virtual “paper” tool and in fact the first system-biological genome browser integrating the holistic complexity of genomes in a single easy comprehensible platform: Based on a detailed study of biophysical and IT requirements, every architectural level from sequence to morphology of one or several genomes can be approached in a real and in a symbolic representation simultaneously and navigated by continuous scale-free zooming within a unique three-dimensional OpenGL and grid driven environment. In principle an unlimited number of multi-dimensional data sets can be visualized, customized in terms of arrangement, shape, colour, and texture etc. as well as accessed and annotated individually or in groups using internal or external data bases/facilities. Any information can be searched and correlated by importing or calculating simple relations in real-time using grid resources. A general correlation and application platform for more complex correlative analysis and a front-end for system-biological simulations both using again the huge capabilities of grid infrastructures is currently under development. Hence, the GLOBE 3D Genome Platform is an example of a grid based approach towards a virtual desktop for genomic work combining the three fundamental distributed resources: i) visual data representation, ii) data access and management, and iii) data analysis and creation. Thus, the GLOBE 3D Genome Platform is the novel system-biology oriented information system urgently needed to access, present, annotate, and to simulate the holistic genome complexity in a unique gateway towards a real understanding, educative presentation and curative manipulation planning of this tremendous evolutionary information grail – genomes

G3DV: A new 3D genome browser and experimental data viewer.

Genomes are tremendous co-evolutionary holistic systems for molecular storage, processing and fabrication of information. Their system-biological complexity remains, however, still largely mysterious, despite immense sequencing achievements and huge advances in the understanding of the general sequential, three-dimensional and regulatory organization. Here, we present the GLOBE 3D Genome Platform a completely novel grid based virtual “paper” tool and in fact the first system-biological genome browser integrating the holistic complexity of genomes in a single easy comprehensible platform: Based on a detailed study of biophysical and IT requirements, every architectural level from sequence to morphology of one or several genomes can be approached in a real and in a symbolic representation simultaneously and navigated by continuous scale-free zooming within a unique three-dimensional OpenGL and grid driven environment. In principle an unlimited number of multi-dimensional data sets can be visualized, customized in terms of arrangement, shape, colour, and texture etc. as well as accessed and annotated individually or in groups using internal or external data bases/facilities. Any information can be searched and correlated by importing or calculating simple relations in real-time using grid resources. A general correlation and application platform for more complex correlative analysis and a front-end for system-biological simulations both using again the huge capabilities of grid infrastructures is currently under development. Hence, the GLOBE 3D Genome Platform is an example of a grid based approach towards a virtual desktop for genomic work combining the three fundamental distributed resources: i) visual data representation, ii) data access and management, and iii) data analysis and creation. Thus, the GLOBE 3D Genome Platform is the novel system-biology oriented information system urgently needed to access, present, annotate, and to simulate the holistic genome complexity in a unique gateway towards a real understanding, educative presentation and curative manipulation planning of this tremendous evolutionary information grail – genomes

A chromosome 21-specific cosmid cocktail for the detection of chromosome 21 aberrations in interphase nuclei

Fluorescent in situ hybridization (FISH) with a 21q11-specific probe (CB21c1) consisting of three non-overlapping cosmids has been applied to interphase amniocytes of pregnancies at increased risk for fetal aneuploidy (N = 78) and to interphase lymphocytes, cultured and uncultured, of patients referred for Down syndrome (N = 19 and 28, respectively). In the uncultured amniocytes, six chromosome aberrations were detected: three cases of trisomy 21, a triploidy, a de novo 46,XX,t(21q21q), and a mosaic 46,XY/47,XY,+dic(21)(q11)/48,XY,+dic(21)(q11), +del(21)(q11). In 15 cultured and 20 uncultured blood samples, FISH correctly diagnosed trisomy 21 (full or mosaic) at the interphase level, which was confirmed in all cases by subsequent karyotyping. Because of specific and strong signals in interphase nuclei, CB21c1 appears to be a useful tool for the rapid detection of chromosome 21 abnormalities

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

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 respecti

Chromatin Dynamics of the mouse β-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 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 down- stream 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. 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, 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. 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. Globin gene activation is depending on long distance looping of the up- and downstream HS sites and the ?-major promotor to the LCR, resulting in a complex 3D chromatin structure. By staining the m?- globin loci with fluorescence labeled sequence specific probes followed by high-resolution 3D imaging and 3D volume rendering of the deconvolved images, the loci reveal changes in the geometric size and shape properties when cells are differentiated into a active globin transcribing cell. An almost 2x decrease in volume was measured, which was mostly due to a reduction of the longest length measured. This can be explained by a change in loop formation. The almost 70Kb loop between the LCR and the 3’HS1 is folded into two loops of 34 and 35Kb upon interaction of the promotor to the ACH to activate transcription. The limited decrease in volume and length when the locus was probed with an additional 5’ and 3’ end region is surprising. The 5’ end is actively participating in the looping process that stabilizes the ACH. However, the 3’ end has (until now) not been seen to be participate in ACH formation or any complex looping mechanism for globin gene activation. As this part of the locus seems to be the most un-dynamic, it could be the dominating factor that influences the fluorescent signal emitting from the probed DNA region and therefore cloud subtle changes in the chromatin folding mechanism of gene activation. Next to the dynamic chromatin folding process that is occurring between the HS-85/84 and the 3’HS1, a stretch of “rigid” DNA can prevent a DNA region containing activated genes to stay at the edge of a chromosome territory and possibly prevent a close proximity to the silencing effect of (spreading) heterochromatin. An increase in lateral and axial resolution like the 3D Structural Illumination Microscope (SIM) provides, could help solve the problem of detecting subtle 3D changes in chromatin structure. And in the near future will reveal many more details of 3D chromatin organization of not only the m?-globin locus but of many other intra-cellular processes

Characterization of a zinc-finger protein and its association with apoptosis in prostate cancer cells

BACKGROUND: The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product. METHODS: The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided. RESULTS: Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells. CONCLUSIONS: We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer

The 3D chromatin structure of the mouse β-haemoglobin gene cluster

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

Chromosomal rearrangements in uveal melanoma: Chromothripsis

Uveal melanoma (UM) is the most common primary intraocular malignancy in the Western world. Recurrent mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, EIF1AX, and SF3B1 are described as well as non-random chromosomal aberrations. Chromothripsis is a rare event in which chromosomes are shattered and rearranged and has been reported in a variety of cancers including UM. SNP arrays of 249 UM from patients who underwent enucleation, biopsy or endoresection were reviewed for the presence of chromothripsis. Chromothripsis was defined as ten or more breakpoints per chromosome involved. Genetic analysis of GNAQ, GNA11, BAP1, SF3B1, and EIF1AX was conducted using Sanger and next-generation sequencing. In addition, immunohistochemistry for BAP1 was performed. Chromothripsis was detected in 7 out of 249 tumors and the affected chromosomes were chromosomes 3, 5, 6, 8, 12, and 13. The mean total of fragments per chromosome was 39.8 (range 12-116). In 1 UM, chromothripsis was present in 2 different chromosomes. GNAQ, GNA11 or CYSLTR2 mutations were present in 6 of these tumors and 5 tumors harbored a BAP1 mutation and/or lacked BAP1 protein expression by immunohistochemistry. Four of these tumors metastasized and for the fifth only short follow-up data are available. One of these metastatic tumors harbored an SF3B1 mutation. No EIF1AX mutations were detected in any of the tumors. To conclude, chromothripsis is a rare event in UM, occurring in 2.8% of samples and without significant association with mutations in any of the common UM driver genes

VACTERL association etiology: The impact of de novo and rare copy number variations

Copy number variations (CNVs), either DNA gains or losses, have been found at common regions throughout the human genome. Most CNVs neither have a pathogenic significance nor result in disease-related phenotypes but, instead, reflect the normal population variance. However, larger CNVs, which often arise de novo, are frequently associated with human disease. A genetic contribution has long been suspected in VACTERL (Vertebral, Anal, Cardiac, TracheoEsophageal fistula, Renal and Limb anomalies) association. The anomalies observed in this association overlap with several monogenetic conditions associated with mutations in specific genes, e.g. Townes Brocks (SALL1), Feingold syndrome (MYCN) or Fanconi anemia. So far VACTERL association has typically been considered a diagnosis of exclusion. Identifying recurrent or de novo genomic variations in individuals with VACTERL association could make it easier to distinguish VACTERL association from other syndromes and could provide insight into disease mechanisms. Sporadically, de novo CNVs associated with VACTERL are described in literature. In addition to this literature review of genomic variation in published VACTERL association patients, we describe CNVs present in 68 VACTERL association patients collected in our institution. De novo variations (>30 kb) are absent in our VACTERL association cohort. However, we identified recurrent rare CNVs which, although inherited, could point to mechanisms or biological processes contributing to this constellation of developmental defects