Commentary 839 IntroductionHuman cells contain 46 chromatin fibres, i.e. the chromosomes, with a total length of ~5 cm of nucleosomal filaments arranged like beads on a string. Packaging this in an interphase nucleus of typically 5-20 m in diameter requires extensive folding. In the past decades, considerable evidence was accumulated showing that chromatin folding is closely related to genome function. Tightly packed and transcriptionally silent heterochromatin, and more-open transcriptionally active euchromatin represent two classic folding states. In the past decade, we started to see some first principles of chromatin folding. One is that individual chromosomes occupy discrete domains in the interphase nucleus -named chromosome territories -which intermingle only to a limited extent (Cremer and Cremer, 2010). Similarly, different parts of a chromosome also only interact very little (Dietzel et al., 1998;Goetze et al., 2007a;Goetze et al., 2007b). Another organisational principle is based on the finding that mammalian interphase chromosomes are made up of a large number of structural domains, each of which are on averagẽ 1 Mb, that correspond to DNA replication units (Ryba et al., 2010). Furthermore, recent experimental data show that chromatin loops mediated by specific chromatin-chromatin interactions are an important aspect of chromatin organisation, because they bring together distant regulatory elements that control gene expression, such as promoters and enhancers (Carter et al., 2002;Kadauke and Blobel, 2009). Methods that are based on the chromosome conformation capture (3C) technology, which determines two distant genomic sequence elements that are in close proximity in the nucleus (Simonis et al., 2007), have revealed the presence of a large number of intra-chromosomal chromatin-chromatin interactions. The resulting loops vary in length from a few kb to up to tens of Mb and are different in different cell types (Lieberman-Aiden et al., 2009;Simonis et al., 2006). Moreover, it has been shown for many loci that changes in transcriptional activity are tightly correlated with changes in folding (Sproul et al., 2005). Together, these observations show that packaging of the chromatin fibre in the interphase nucleus is closely related to genome function and that loops are a prominent feature of interphase chromatin.The notion that chromatin loops are important for overall genome organisation is also supported from the perspective of polymer models. Recent polymer modelling efforts show that the formation of loops can endow polymers such as chromatin with properties that explain several of its key properties, including chromatin compaction and compartmentalisation. The importance of polymer models is that they aim to explain properties of chromatin on the basis of physical principles and discard those models that do not fulfil this criterion. They make precise predictions that can be tested experimentally and their outcome can be used to further improve the model, thereby increasing our understanding of chro...

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The epigenome is a heritable layer of information not encoded in the DNA sequence of the genome, but in chemical modifications of DNA or histones. These chemical modifications, together with transcription factors, operate as spatiotemporal regulators of genome activity. Dissecting epigenome function requires controlled site-specific alteration of epigenetic information. Such control can be obtained using designed DNA-binding platforms associated with effector domains to function as targeted transcription factors or epigenetic modifiers. Here, we review the use of dCas9 as a novel and versatile tool for fundamental studies on epigenetic landscapes, chromatin structure and transcription regulation, and the potential of this approach in basic research in these fields.

Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type–specific transcription. We know remarkably little about the principles that control chromosome folding. Here we explore the view that intrachromosomal interactions, forming a complex pattern of loops, are a key element in chromosome folding. CTCF and cohesin are two abundant looping proteins of interphase chromosomes of higher eukaryotes. To investigate the role of looping in large-scale (supra Mb) folding of human chromosomes, we knocked down the gene that codes for CTCF and the one coding for Rad21, an essential subunit of cohesin. We measured the effect on chromosome folding using systematic 3D fluorescent in situ hybridization (FISH). Results show that chromatin becomes more compact after reducing the concentration of these two looping proteins. The molecular basis for this counter-intuitive behaviour is explored by polymer modelling usingy the Dynamic Loop model (Bohn M, Heermann DW (2010) Diffusion-driven looping provides a consistent framework for chromatin organization. PLoS ONE 5: e12218.). We show that compaction can be explained by selectively decreasing the number of short-range loops, leaving long-range looping unchanged. In support of this model prediction it has recently been shown by others that CTCF and cohesin indeed are responsible primarily for short-range looping. Our results suggest that the local and the overall changes in of chromosome structure are controlled by a delicate balance between short-range and long-range loops, allowing easy switching between, for instance, open and more compact chromatin states.

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Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project toward the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.