Final Report Summary - 4C (4C technology: uncovering the multi-dimensional structure of the genome)
At the time of applying for an ERC Starting grant, the de Laat research group had just developed an exciting new technology for the genome-wide assessment of DNA contacts made a genomic site of choice. DNA contacts between gene promoters and regulatory sequences such an enhancer, the positioning of genes in the nuclear interior relative to each other, to the periphery and perhaps to other nuclear landmarks were all emerging as important epigenetic contributors to the genome functioning. Our methodology, named 4C technology, promised to be very instrumental in uncovering the relationship between genome structure and function and the proposal therefore centered around the further development and application of 4C technology.
Our research led to exciting discoveries and the development of novel methodologies. We investigated if and how DNA topology changes during cellular differentiation and showed that the 3D pluripotent genome is uniquely shaped around pluripotency factors, with inactive chromatin adopting random nuclear positions being naively shaped in embryonic cells (de Wit et al., Nature 2013). Thus, tissue-specific transcription factors fold their genomes, and a naïve chromatin organization may help confer plasticity to stem cells. To understand whether functional crosstalk may exist also between chromosomes that contact each other in the nuclear space, we created a unique set of transgenic mice carrying a new super enhancer at a defined chromosomal location. We provided first genetic evidence for trans-activation in mammals: an enhancer on one chromosome can activate a gene on another chromosome. This work had further interesting implications as it showed that cell-specific genome conformations can cause variegated gene expression (i.e. single cells among otherwise identical cells express a given gene at very different levels. A further major observation was the demonstration that RNA can shape the genome: we showed that the inactive X chromosome adopts a unique 3D structure that is dependent on Xist RNA.
Technological innovations included the development of next generation sequencing based strategies for the analysis of DNA contacts, with high resolution 4C-seq as a technique giving superior resolution DNA contact profiles and being the method of choice for the detection of regulatory sequences controlling the expression of any gene of interest. This method is now widely used in the field that studies the relationship between gene structure and function. Another major technological breakthrough was the demonstration that chromosome conformation capture technologies, originally designed to study the 3D genome, are also very instrumental for the analysis of the linear genome, and therefore for the analysis of chromosomal rearrangements. Application of this method resulted in the discovery of multiple new oncogenes involved in cancer, and laid the foundation of yet other new technologies that give superior complete sequence analysis of genomic regions of choice and that are finding their ways into the clinic.
Our research led to exciting discoveries and the development of novel methodologies. We investigated if and how DNA topology changes during cellular differentiation and showed that the 3D pluripotent genome is uniquely shaped around pluripotency factors, with inactive chromatin adopting random nuclear positions being naively shaped in embryonic cells (de Wit et al., Nature 2013). Thus, tissue-specific transcription factors fold their genomes, and a naïve chromatin organization may help confer plasticity to stem cells. To understand whether functional crosstalk may exist also between chromosomes that contact each other in the nuclear space, we created a unique set of transgenic mice carrying a new super enhancer at a defined chromosomal location. We provided first genetic evidence for trans-activation in mammals: an enhancer on one chromosome can activate a gene on another chromosome. This work had further interesting implications as it showed that cell-specific genome conformations can cause variegated gene expression (i.e. single cells among otherwise identical cells express a given gene at very different levels. A further major observation was the demonstration that RNA can shape the genome: we showed that the inactive X chromosome adopts a unique 3D structure that is dependent on Xist RNA.
Technological innovations included the development of next generation sequencing based strategies for the analysis of DNA contacts, with high resolution 4C-seq as a technique giving superior resolution DNA contact profiles and being the method of choice for the detection of regulatory sequences controlling the expression of any gene of interest. This method is now widely used in the field that studies the relationship between gene structure and function. Another major technological breakthrough was the demonstration that chromosome conformation capture technologies, originally designed to study the 3D genome, are also very instrumental for the analysis of the linear genome, and therefore for the analysis of chromosomal rearrangements. Application of this method resulted in the discovery of multiple new oncogenes involved in cancer, and laid the foundation of yet other new technologies that give superior complete sequence analysis of genomic regions of choice and that are finding their ways into the clinic.