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Biomedical genomics
Genome structure and function in health and disease

Nuclear organisation and gene expression
The architecture of DNA in the interior of the living cell nucleus is an emerging key contributor to genomic function. For example, we, and others, have shown that transcription regulatory DNA elements loop out intervening DNA to contact distant target genes and regulate their expression in vivo. Moreover, individual genes may move to other nuclear locations upon changes in their transcription status. Besides transcription, genomic organization is associated with the coordination of replication, recombination and the probability of loci to translocate (which can lead to malignancies) and the setting and resetting of epigenetic programs. 
Aim of our research is to understand the relationship between genome structure and function in mammalian cells.
Techniques We use and develop novel genomics approaches (next generation sequencing and microarrays) and bioinformatic analysis tools, in combination with high-resolution microscopy techniques and molecular and cell biology strategies. We recently developed 4C technology, a novel high-throughput genomics approach that uniquely allows screening the entire genome for DNA regions that interact with a DNA segment of choice. 4C is expected to importantly contribute to our understanding of nuclear architecture.
Our previous work In the recent past we have investigated how transcription regulatory DNA elements communicate with genes over distance. For this, we have adapted 3C technology and applied it to investigate the conformation of the mammalian beta-globin gene locus. We demonstrated that distant transcription regulatory DNA elements physically contact their target genes for gene regulation, with intervening chromatin looping out. In fact, multiple transcription regulatory DNA elements and active genes were found to spatially cluster and form a so-called Active Chromatin Hub (ACH), being a chromatin structure that facilitates efficient transcription in vivo (Tolhuis et al., Mol Cell 2002 ). We subsequently showed that the DNA topology of a gene locus changes during development and differentiation ( Palstra et al., Nat Genet 2003 ). We also showed that transcription factors set up the three-dimensional chromatin structure of gene loci in vivo (Drissen et al., Genes Dev 2004; Splinter et al., Genes Dev 2006; Kooren et al., JBC 2007 ) (fig. 1).

More recently we have developed 4C technology, a gnomics approach that uniquely allows for the unbiased genome-wide screening of DNA elements that contact a locus of choice in the nuclear space. 4C technology is comparable to chromatin immunoprecipitation-on-chip (ChIP-on-chip), but interrogates DNA-DNA, instead of DNA -protein interactions (fig. 2).

Using 4C technology, we provided the first comprehensive pictures of long-range intra- and interchromosomal contacts of gene loci in living cells. We demonstrated that a tissue-specific gene switches its nuclear environment in relation to its expression status, and showed that long-range intra- and interchromosomal contacts with a housekeeping gene are conserved between tissues (Simonis et al. Nat Genet, 2006) (fig. 3).

To verify and further investigate the intra- and interchromosomal contacts identified by 4C, we have set up high-resolution cryo-FISH (fig. 4). These microscopy experiments consistently show that 4C technology robustly identifies long-range DNA contacts formed in the nucleus.

Our current research is focusing on the further development and application of 4C technology in order to get a comprehensive understanding of the functional significance of nuclear architecture, follow chromosome folding during cellular differentiation and uncover the proteins that dictate the shape of our genome.

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Figure 1. the beta-globin Active Chromatin Hub (ACH) during development and erythroid differentiation and the role of the transcription factors CTCF and EKLF in the establishment of the ACH.


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Figure 2. 4C technology



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Figure 3. long-range interactions with the beta-globin locus. Depicted are beta-globin interacting regions in cis (on chr. 7) in fetal liver (red; beta-globin active) and fetal brain (blue; inactive).



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Figure 4. Cryo-FISH. A, Image with multiple cells, note that due to sectioning, only some cells show a FISH signal. Examples of loci scored as contacting (b-c) and non-contacting (d-f).