Chromosome Research (2008) 16:397–412 # Springer 2008 DOI: 10.1007/s10577-008-1237-3

Elucidating and nuclear domain architecture with electron spectroscopic imaging

David P. Bazett-Jones1*,RenLi1, Eden Fussner1, Rosa Nisman1 & Hesam Dehghani2 1Program in Genetics and Genome Biology, The Hospital for Sick Children, Research Institute, 101 College Street, East Tower, 15th Floor, 15-401T, Toronto, ON M5G 1L7, Canada; Tel: +1-416-813-2181; E-mail: [email protected]; 2Department of Physiology, School of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran The authors dedicate this paper to the memory of Ying Ren (1961Y2007). We all benefited from knowing her. Our research advanced through the technical creativity she provided. *Correspondence

Key words: chromatin, correlative microscopy, electron microscopy, heterochromatin, nuclear speckles, nuclear structure, , promyelocytic leukemia, transcription

Abstract

Electron microscopy has been the Fgold standard_ of spatial resolution for studying the structure of the nucleus. Electron spectroscopic imaging (ESI) offers advantages over conventional transmission electron microscopy by eliminating the need for heavy-atom contrast agents. ESI also provides mass-dependent and element-specific information at high resolution, permitting the distinguishing of structures that are primarily composed of protein, DNA, or RNA. The technique can be applied to understand the structural consequences of epigenetic modifications, such as modified histones, on chromatin fiber morphology. ESI can also be applied to elucidate the multifunctional behavior of subnuclear Forganelles_ such as the nucleolus and promyelocytic leukemia .

Abbreviations SR serine-arginine SUMOylation small -like modifier post-translational CBP Creb-binding protein modification CC condensed chromatin CTEM conventional transmission electron microscopy Introduction Daxx death-associated protein 6 ES murine embryonic stem cell ESI electron spectroscopic imaging The is composed of several compart- GFP green fluorescent protein ments including chromosome territories, nucleoli, ICS interchromosome space nuclear speckles (interchromatin granule clusters), IGC interchromatin granule clusters replication and transcription factories, promyelocytic ING1 inhibitor of growth family member 1 leukemia nuclear bodies (PML NBs), and a number MEL murine erythroleukemia cell Mdm2 murine double minute protein 2 of other bodies and assemblies. All await further PML NB promyelocytic leukemia nuclear body analysis and functional characterization. How these Rb retinoblastoma structures within the nucleus are organized appears to 398 D. P. Bazett-Jones et al. be largely a function of the various nuclear activities, energy losses that arise from core loss ionizations are such as transcription or replication of DNA (Kosak & discrete and are element-specific. The incident elec- Groudine 2004), yet the fundamental rules governing trons, which cause these events, could therefore be the relationship of nuclear structure to function used to generate element-specific images if it were remain to be elucidated. possible to filter or select these electrons from the Nuclear compartments are assembled and main- electron energy loss spectrum. Electron imaging spec- tained in a dynamic fashion (Misteli 2001), likely trometers have been designed and implemented for dictated by their function and involvement in nuclear this purpose. These devices accomplish two functions. processes. Nucleolar organization, for example, pro- First, in functioning as an electron spectrometer, they vides a unique spatial clustering of ribosomal genes separate electrons according to their energy losses. from d