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Towards the Visualization of Genome Activity at Nanoscale Dimensions University of Massachusetts Medical School eScholarship@UMMS Open Access Articles Open Access Publications by UMMS Authors 2006-03-07 Towards the visualization of genome activity at nanoscale dimensions Joan C. Ritland Politz University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/oapubs Part of the Biochemistry, Biophysics, and Structural Biology Commons, and the Genomics Commons Repository Citation Politz JC. (2006). Towards the visualization of genome activity at nanoscale dimensions. Open Access Articles. https://doi.org/10.1186/gb-2006-7-1-304. Retrieved from https://escholarship.umassmed.edu/ oapubs/606 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in Open Access Articles by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. Meeting report Towards the visualization of genome activity at nanoscale comment dimensions Joan C Ritland Politz Address: Program in Cell Dynamics, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 377 Plantation Street, Worcester, MA 01605, USA. Email: [email protected] Published: 1 February 2006 reviews Genome Biology 2006, 7:304 (doi:10.1186/gb-2006-7-1-304) The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2006/7/1/304 © 2006 BioMed Central Ltd reports Park, USA) discussed new algorithms designed to identify A report on the Fifth Annual Nanostructural Genomics important genomic regions that may not be coding sequence meeting, Bar Harbor, USA, 7-10 September 2005. but are nevertheless conserved between organisms. These algorithms, including phastCons and RP (regulatory poten- tial), use methodology such as alphabet clustering, where It is a rare meeting where one can hear the latest develop- different nucleotide-sequence patterns are each classified as deposited research ments in comparative genome analysis, relate these findings a letter of the alphabet, to reduce complexity and identify to advances in understanding both the linear and three- higher-order sequence patterns that may be conserved ‘in dimensional organization of the eukaryotic genome, and see it spirit’, if not in exact sequence, in the genome. Some algo- all beginning to fit into the context of the structure and func- rithms are better than others at identifying particular tion of the nucleus, visualized using state-of-the art labeling sequence features; for example, phastCons identifies poten- and microscopic techniques. These cross-disciplinary areas of tial microRNA genes better than RP. research have been presented by a diverse group of scientists for the past five years at the Nanostructural Genomics meeting Moving to the next organizational level, chromatin, Jason at the Jackson Laboratory in Bar Harbor, and the 2005 Lieb (University of North Carolina, Chapel Hill, USA) refereed research meeting again gave attendees much food for thought. described a novel approach to the study of the structure of active chromatin in yeast. Using chromatin immunoprecipi- In his opening address, Timothy O’Brien (Cornell University, tation (ChIP) he has compared the pattern of sites identified Ithaca, USA) outlined his view of how genomics, cell biology by binding in vivo of the DNA-binding domain of the tran- and optical physics all work together to create an accurate scription factor Leu3 to the pattern obtained by ‘DIP ChIP’, picture of nuclear structure and function, which can lead to in which naked DNA is allowed to bind the Leu3 protein in important insights into cellular form and function. He dis- vitro and is then crosslinked and immunoprecipitated. By cussed his studies of a several megabase region surrounding comparing the two experiments he found that promoters interactions the mouse piebald locus, a genetically defined region named contain fewer nucleosomes than do other DNA sites. In addi- after a coat-color gene within it. He used comparative tion, Lieb showed that, even at the promoter, nucleosomal genomics to learn more about the nature of particular dele- organization is dynamic and influences the type of protein tions in this region that cause neonatal respiratory distress that binds to a particular site. Evidence of promotor-specific and death. This information was coupled to high-resolution chromatin structure in the human genome came from Keji visualization of gene-rich and gene-poor sections of this Zhao (National Heart, Lung and Blood Institute, National region in the nucleus, and to the prediction of potential tran- Institutes of Health, Bethesda, USA), who used ChIP in com- scription-factor binding sites for specific genes, such as bination with serial analysis of gene expression (SAGE) to information sprouty2, a gene involved in lung branching morphogenesis. show that histone-acetylation islands in the human genome correlate with active promoter regions but not with the entire transcriptionally active gene. Chromosome sequence and structure Considering comparative genomics at the sequence level, The notion that the three-dimensional organization of chro- Ross Hardison (Pennsylvania State University, University matin reflects gene activity is intellectually satisfying but has Genome Biology 2006, 7:304 304.2 Genome Biology 2006, Volume 7, Issue 1, Article 304 Politz http://genomebiology.com/2006/7/1/304 not yet been rigorously proven. Roel van Driel (University of Biophysical Chemistry, Göttingen, Germany) described a Amsterdam, The Netherlands) described a comprehensive stimulated emission depletion (STED) light microscope study that is designed to determine whether gene-rich system in which the Abbe diffraction resolution limit (the regions, which tend to be clustered on the linear map in usual limit of a light microscope) has been broken. This has ‘ridges’ (regions of increased gene expression), occupy dis- been achieved by inhibiting the fluorescence of molecules at tinct nuclear domains. Using fluorescent in situ hybridiza- the outer region of a scanning excitation spot in a saturated tion (FISH) to tag different genomic regions in a systematic manner. With the use of carefully chosen dyes and focal way, he and his collaborators have found that, despite sur- intensity conditions, Hell has attained 10 nm optical resolu- prisingly large cell-to-cell variations, on average gene-rich tion (in the lateral x-y dimension). The 4Pi microscope from and gene-poor regions seldom overlap spatially in the inter- Leica Microsystems, an application of Hell’s earlier ideas, was phase nuclei of primary human fibroblasts or HeLa cells. also demonstrated at the meeting by Lindsay Shopland (The Their initial studies also suggest that different gene-rich Jackson Laboratory) and Joerg Bewersdorff (The Jackson regions themselves might occupy non-overlapping territo- Laboratory). This system increases optical resolution in the ries within the nucleus (and gene-poor regions also appear axial dimension (z-dimension) by the use of two opposing to be within distinct territories). O’Brien presented data on objective lenses to propagate light from multiple directions this topic from the piebald locus, showing images (obtained toward the focal point, followed by a deconvolution step to by Lindsay Shopland, Jackson Laboratory, Bar Harbor, give a z-resolution of about 80 nm (this is about five- to USA) of chromatin hybridized in situ with differently colored sevenfold greater than conventional light microscopy). This is fluorescent bacterial artificial chromosomes complementary about the size of an average gene domain. Both these to either gene-rich or gene-poor regions of the piebald locus. systems increase optical resolution (the smallest distance In some cases, the gene-rich regions were clustered together detectable between two small objects) and thus also struc- in ‘hubs’, which can be loosely defined as congregations of tural visualization and distinction to levels that have up to regulatory and/or transcriptionally active genes that are not now been impossible to reach with light microscopy, and necessarily adjacent on the linear genome. In other cells, span the 100 nm region that has classically been above the however, the gene-rich and gene-poor regions remained practical range of electron microscopy and below that of interspersed linearly along the piebald region, giving the light microscopy. chromosome a ‘candy cane’ appearance. Advances in electron microscopy were not neglected. David Job Dekker (University of Massachusetts Medical School, Bazett-Jones (The Hospital for Sick Children, Toronto, Worcester, USA) came at the question of the three-dimen- Canada) discussed the use of electron spectroscopic imaging sional organization of active versus inactive chromatin from a to study the structure and composition of intranuclear more biochemical angle, using his previously published bodies at the nanometer level. This technique takes advan- method of chromosome conformation capture (3C). He found tage of the fact that electrons lose differing amounts of that actively transcribing regions of the ␤-globin locus, which energy depending on which elements they excite or ionize form decondensed ‘puffs’, are near one another (crosslink- when they pass through a sample. Using this extremely able) in hubs, whereas the more compact, repressed chro- informative technique, Bazett-Jones learned that subnuclear matin
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