Hi-C maps of the genome quantitatively reflect the interactions of the nucleotides within the analyzed regions of the genome. All contact points are represented graphically on heat maps (see figure), where the intensity or rather the number of contacts is specified on a red color scale.
Hi-C is used to analyze the spatial organization of the DNA inside the nucleus by quantifying the number of interactions between genomic loci that are nearby in 3-D space, but may be separated by many nucleotides on the linear genome. To find out more about this, they investigated the DNA of the limb buds of the modified mouse embryos with a set of methods called Hi-C. The scientists were particularly interested in which elements of the DNA interact with each other. Its activation before day 12.5 is known to cause polydactyly." “Ihh is important for the formation of the limbs, but physiologically, it is expressed later in development. "We believe that this is due to the activation of the Ihh gene, which belongs to the genes that have come into the scope of action of the enhancer through the inversion," explains Mundlos. The mice with inversions 1 and 2 also showed a polydactyly, that is, they developed more than five toes on their paws. The researchers could show that the inversions induced an activation of the genes, which had been positioned to Epha4 enhancer vicinity by the inversion, at day 11.5. In wild type embryos, the Epha4 gene is active during this time, while the other four genes are inactive. The team focused on day 11.5 of mouse development. Each inversion ended individually at the promoters of four different genes in the gene-dense region. All inversions started at the same point of the DNA and included an enhancer within the Epha4 TAD, which is important for limb development, as well as the CTCF element at this end of the TAD. For this, they cut four segments of different length out of the DNA and re-inserted them at the same place, but in opposite direction (inverted). The scientists analyzed the functional effects of genomic inversions in this region. Inversions influence timing of gene expression In contrast, a gene-dense region between the Epha4 and Pinc TADs, shows no clear structure. Analysis of the 3D structure of the region showed, that three genes ( Epha4, Pax3, Pinc) reside in clearly defined TADs. This is a region in the genome that contains many developmentally important genes. Now, together with scientists from the Department of Computational Molecular Biology, also at MPIMG, his team has studied the Epha4 locus. In recent years, he has studied the formation of TADs to find out what happens, when congenital rearrangements break the architectural configuration, e.g, shift the defined boundary of a TAD. He is particularly interested in how changes in the genome can cause congenital malformations or diseases such as cancer. "In addition to the TADs, we also find gene-dense regions in the genome where no clear TAD structures can be discerned." Mundlos is a human geneticist. "But not all regions of the genome are organized in distinct TADs," explains Stefan Mundlos, head of the research group Development & Disease at the Max Planck Institute for Molecular Genetics in Berlin. If the cohesin comes from the opposite side or the CTCF element is inverted, the cohesin ring slides over it without any stop function at the respective position. But this mechanism functions only in one direction. They are formed by ring-shaped protein-complexes called cohesin, which extrude a DNA loop by translocating along the DNA strand in both directions until they are stopped by the CTCF elements representing the boundaries of the TADs. TADs have a clear structure with defined boundaries, which insulate the regulatory activity within the TAD and define the genes an enhancer can act on. These are "loops" in the genome containing one or a few genes and their regulatory elements (enhancers, promoters). Formations fairly well understood are the TADs (topologically associated domains). But the three-dimensional structure of DNA also plays an important role. They usually bind other molecules (transcription factors) that are required for reading the genes. Well-known regulators include enhancers or promoters, specific regions within the DNA with regulatory function. So how does a cell know, at what time during embryonic development it should grow and what tissue it should develop? Crucial for the differentiation of the cells is the gene activity a process controlled by many different mechanisms and at different levels. But a thumb looks different from an index finger a hand looks different from a foot. Each cell of an organism contains the organism's complete DNA.
0 kommentar(er)
