Keeping the genome in shape : a role for protein and RNA

Abstract

Over 200 cell types exist within the human body, each being different in morphology and function, yet all containing the same genome. Regulation programs acting on the approximately 25.000 genes found in a typical mammalian genome drive the specification of cells during development. Also during later stages of life correct gene expression is required, as defects can lead to diseases such as cancer. To ensure correct spatial and temporal expression of genes different control mechanisms are in place acting on different levels of gene regulation. Gene regulatory information is primarily encoded in the DNA sequence. However, over the past decade also the three dimensional folding of the DNA fiber inside the nucleus has been implicated in regulating gene expression. To investigate this we performed experiments aimed at unraveling the shape of the genome, studying the relationship between genome structure and function and identifying the factors responsible for genome folding. Part of the research conducted focused on the development and adaptation of a novel technology called ‘Chromosome Conformation Capture’ (3C) to investigate nuclear organization. 3C allows one to interrogate the frequency by which specific DNA fragments contacted each other in nuclear space. When measuring these frequencies for multiple combinations of fragments this technology provides detailed information on the spatial organization of a gene locus. Developing this technology one step further to a technology named ‘4C’, allowed us to investigate the spatial environment of a given DNA fragment by analyzing all the contacting DNA fragments simultaneously. Both 3C and 4C methods were applied on the mouse β-globin locus. In this study, both by using cells carrying conditional CTCF alleles and taking advantage of cells carrying a targeted mutation of a CTCF site within the globin locus, we investigated the role of CTCF in dictating chromatin interactions. This study provides novel insights into the role of CTCF in mediating loop formation. Moreover, the effect of these loops in dictating spatial contacts and in facilitating correct gene expression in the β-globin locus was examined. Next we switch from investigating locus wide conformations, to interrogating the spatial organization of a whole chromosome. The spatial organization of both the active and inactive female mouse X-chromosome was determined. To be able to discriminate between the active and inactive X-chromosome in female mammalian cells we developed an allele specific 4C-seq approach. Based on the presence of single nucleotide polymorphisms (SNPs), this method enabled separating the contacts made by the two chromosomes. Moreover, by studying cells depleted for the noncoding RNA Xist, this method was used to elucidate the role of this RNA molecule in dictating chromosome conformation. Overall detailed information is provided concerning the 3C and 4C methods that were used to investigate the spatial organization of DNA inside the nucleus. The studies performed ranged from investigating locus wide to chromosome wide spatial organization. Moreover, the protein CTCF and the noncoding Xist RNA were identified as factors involved in chromatin folding, each acting at a different level of organization, and providing novel insights into the mechanisms by which DNA is organized inside the nucleus of a cell. Keywords Genome organization