de Laat: Biomedical genomics

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The De Laat group studies genome structure and function in development and disease and develops DNA sequencing methods for improved clinical diagnostics.

Nuclear organisation and gene expression

The mammalian genome contains millions of non-coding sequences with transcriptional regulatory potential, including enhancers, which can activate gene expression, and insulators, which can block enhancer action. A main interest of our work is to understand how this dizzying array of regulatory sequences is functionally wired to the roughly twenty thousand genes present, such that the orchestration of gene expression is properly executed in both time and place, and how faulty wiring of these elements leads to disease.

Thanks to the work of many researchers, including those from our own group, the shape of the genome is now appreciated as an important contributor to transcriptional control. Chromosome conformation capture (3C) technologies in particular have greatly contributed to this understanding and have uncovered a hierarchical organization of genome topology that is intimately linked to the regulation of gene expression. Enhancers act on (distal) target genes via 3D chromatin looping. They function in the spatial context of structural domains or ’topologically associating domains’ (TADs), which are believed to demarcate microenvironments for genes and regulatory elements to roam within and to make productive DNA contacts. In the larger nuclear space, TADs segregate and cluster into active (A) and inactive (B) compartments that can each be further divided into nuclear sub-compartments with distinct chromatin signatures.

Hierarchical 3D genome organisation, showing chromosome territories (left), nuclear A and B compartments, TADs and and sub-TADs (middle panel) and chromatin loops (right panels). Adapted from Denker and de Laat, Genes Dev 2016.

The aim of our research is to deepen our understanding of the relationship between genome structure and function in mammalian cells, in health and in disease.

4C versus Hi-C

Techniques

We use and develop novel genomics approaches and computational tools that employ Next Generation Sequencing and Oxford Nanopore Single Molecule Sequencing (a) to accelerate progress in our understanding of the 3D genome and its impact on gene regulation and (b) for improved DNA diagnostics in the clinic. As an illustration:

  • We were the first to adapt 3C technology to study enhancer-promoter interactions (Tolhuis, 2002)
  • We developed 4C technology (Simonis, 2006)
  • We developed high-resolution 4C technology for detailed DNA contact studies focused on genes and regulatory sequences (van de Werken, 2013)
  • We demonstrated that ‘C’ technologies can be employed as diagnostic tools, for the detection of chromosomal rearrangements (Simonis, 2009)
  • Together with our spin-off company Cergentis we developed Targeted Locus Amplification (TLA technology: de Vree, 2014)
  • We developed multi-contact 4C (Allahyar, 2017)
Strategy for Monogenic Non-Invasive Prenatal Diagnosis. A. Summary of the MG-NIPD approach. Blood is isolated from both parents and cells are used to TLA haplotype the disease locus. Cell-free DNA is isolated from maternal plasma during pregnancy and sequenced to analyze cell-free fetal DNA. Parental locus-specific haplotypes are used to discern which combination of parental alleles is overrepresented in cell-free DNA and therefore inherited by the fetus. B. Targeted haplotyping by TLA. Crosslinking (blue ovals), digestion and proximity ligation primarily yields intra-chromosomal ligation products. Ligation products containing a (viewpoint) SNP of interest (yellow and green triangles) can be selectively amplified by inverse PCR and sequenced. Variants ending up in the same ligation product (indicated respectively by blue and red triangles) are assigned to the same allele (phasing).

Our previous work

Most of our research has been dedicated to understanding gene regulation in development and disease (e.g. cancer) and how this is controlled by non-coding regulatory DNA elements that often lie away from genes on the linear chromosome. In our earlier work, we were the first to adapt 3C technology to study DNA topology in mammals and demonstrate that distant enhancers and promoters contact each other for gene regulation (Tolhuis, Mol Cell 2002; Palstra, Nat Genetics 2003). We also provided first evidence that transcription factors mediate chromatin loops (Drissen, Genes and Dev 2004). In 2006 we were the first to demonstrate that CTCF forms chromatin loops (Splinter, Genes and Dev 2006) and we published our now widely used 4C technology (Simonis, Nat Genetics 2006).

The dynamic beta-globin Active Chromatin Hub (Tolhuis et al., Mol Cell 2002 and Palstra et al., Nat Genetics 2003)

More recent important contributions to this field (last 10 years), include:

  • We provided first evidence that non-coding RNA molecules can change chromosome topology (Splinter et al., Genes and Dev 2011).
  • We provided first genetic evidence in mammals that an enhancer can trans-activate a gene on another chromosome and that cell-specific 3D genomes can contribute to transcriptional noise (Noordermeer, Nat Cell Biol 2011).
  • We showed that ES cells adopt a unique genomic conformation organized around pluripotency factors (de Wit, Nature 2013).
  • We identified PADs (pericentromeric associated domains) and showed their high similarity to LADs (lamin associated domains) (Wijchers, Mol Cell 2016)
  • We demonstrated that the DNA binding polarity of CTCF is important for chromatin loop formation (de Wit, Mol Cell 2015).

A distinctive feature of our research is that in addition to our basic studies on gene regulation in the 3D genome we explore ways to bring our expertise and technologies to the clinic. Highlights:

  • We were the first to demonstrate that 3C technologies are very suitable for the identification of structural chromosomal rearrangements (Simonis, Nat Methods 2009; Homminga, Cancer Cell 2011).
  • With Cergentis, our spin-off company, we developed TLA technology, a novel proximity-ligation based method for robust gene sequencing, which we showed identifies the complete spectrum of genetic variation, including previously missed mutations in cancer genes (de Vree, Nat Biotech 2014).
  • We employed TLA’s targeted haplotyping capacity to develop a sensitive and robust non-invasive prenatal diagnostic test for monogenic diseases (Vermeulen, AJHG 2017).

In summary, our work has given important insight into genome structure and gene regulation and has delivered widely-used genomics tools to the research community. We further successfully pioneer the application of new technologies in clinical diagnostics, which led to the foundation of biotech company Cergentis.