Chromatin movement visualized with photoactivable GFP-labeled histone H4.

The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

[1]  James G. McNally,et al.  Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks , 2006, The Journal of cell biology.

[2]  J. R. Coleman,et al.  Processing of Endogenous Pre-mRNAs in Association with SC-35 Domains Is Gene Specific , 1999, The Journal of cell biology.

[3]  G. Dellaire,et al.  Chromatin Contributes to Structural Integrity of Promyelocytic Leukemia Bodies through a SUMO-1-independent Mechanism* , 2004, Journal of Biological Chemistry.

[4]  Anne E Carpenter,et al.  Long-Range Directional Movement of an Interphase Chromosome Site , 2006, Current Biology.

[5]  Juliet A. Ellis,et al.  The spatial organization of human chromosomes within the nuclei of normal and emerin-mutant cells. , 2001, Human molecular genetics.

[6]  François-Michel Boisvert,et al.  Promyelocytic Leukemia (Pml) Nuclear Bodies Are Protein Structures That Do Not Accumulate RNA , 2000, The Journal of cell biology.

[7]  Paul S. Freemont,et al.  Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions , 2004, The Journal of cell biology.

[8]  H. Tanke,et al.  Advances in fluorescent tracking of nucleic acids in living cells. , 2006, BioTechniques.

[9]  M. Vázquez,et al.  Chromosomes are predominantly located randomly with respect to each other in interphase human cells , 2002, The Journal of cell biology.

[10]  J. Mcneil,et al.  Tracking COL1A1 RNA in osteogenesis imperfecta. splice-defective transcripts initiate transport from the gene but are retained within the SC35 domain. , 2000 .

[11]  Thomas Cremer,et al.  Chromosome territories--a functional nuclear landscape. , 2006, Current opinion in cell biology.

[12]  J. Lawrence,et al.  Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact. , 2001, Molecular biology of the cell.

[13]  F. O. Fackelmayer,et al.  Local Chromatin Mobility is Independent of Transcriptional Activity , 2006, Cell cycle.

[14]  A. von Mikecz,et al.  Cell cycle-dependent association of PML bodies with sites of active transcription in nuclei of mammalian cells. , 2002, Journal of structural biology.

[15]  H. Tanke,et al.  Poly(A)+ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells , 2004, The Journal of cell biology.

[16]  Jan Koster,et al.  The Three-Dimensional Structure of Human Interphase Chromosomes Is Related to the Transcriptome Map , 2007, Molecular and Cellular Biology.

[17]  Alberto Diaspro,et al.  Two-photon activation and excitation properties of PA-GFP in the 720-920-nm region. , 2005, Biophysical journal.

[18]  J. Ragoussis,et al.  Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. , 2000, Journal of cell science.

[19]  H. Leonhardt,et al.  Dynamics of DNA Replication Factories in Living Cells , 2000, The Journal of cell biology.

[20]  A. Pombo,et al.  Intermingling of Chromosome Territories in Interphase Suggests Role in Translocations and Transcription-Dependent Associations , 2006, PLoS biology.

[21]  R. Evans,et al.  Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. van Driel,et al.  The eukaryotic genome: a system regulated at different hierarchical levels , 2003, Journal of Cell Science.

[23]  Peter Teague,et al.  Differences in the Localization and Morphology of Chromosomes in the Human Nucleus , 1999, The Journal of cell biology.

[24]  Mark Groudine,et al.  A Functional Enhancer Suppresses Silencing of a Transgene and Prevents Its Localization Close to Centromeric Heterochromatin , 1999, Cell.

[25]  H. Tanke,et al.  Mobile foci of Sp100 do not contain PML: PML bodies are immobile but PML and Sp100 proteins are not. , 2002, Journal of structural biology.

[26]  S. Gasser,et al.  Visualizing Chromatin Dynamics in Interphase Nuclei , 2002, Science.

[27]  M. C. Butler,et al.  The replication timing program of the Chinese hamster β-globin locus is established coincident with its repositioning near peripheral heterochromatin in early G1 phase , 2001, The Journal of cell biology.

[28]  A. Matera,et al.  RNA-mediated interaction of Cajal bodies and U2 snRNA genes , 2001, The Journal of cell biology.

[29]  Wendy A. Bickmore,et al.  Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH , 2002, The Journal of cell biology.

[30]  Bernd Rieger,et al.  One‐ and two‐photon photoactivation of a paGFP‐fusion protein in live Drosophila embryos , 2005, FEBS letters.

[31]  Cameron S. Osborne,et al.  Replication and transcription: Shaping the landscape of the genome , 2005, Nature Reviews Genetics.

[32]  T. Cremer,et al.  Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions , 2007, Nature Reviews Genetics.

[33]  Thomas Cremer,et al.  Higher order chromatin architecture in the cell nucleus: on the way from structure to function , 2004, Biology of the cell.

[34]  A. Fisher,et al.  Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. , 1999, Molecular cell.

[35]  T. Misteli,et al.  Spatial genome organization during T-cell differentiation , 2004, Cytogenetic and Genome Research.

[36]  Daniel Axelrod,et al.  Chromatin Dynamics in Interphase Nuclei and Its Implications for Nuclear Structure , 1997, The Journal of cell biology.

[37]  J. Lawrence,et al.  Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for PreU2 within Cajal bodies. , 2000, Molecular biology of the cell.

[38]  W. Bickmore,et al.  Re-modelling of nuclear architecture in quiescent and senescent human fibroblasts , 2000, Current Biology.

[39]  R. Sternglanz,et al.  Perinuclear localization of chromatin facilitates transcriptional silencing , 1998, Nature.

[40]  Hiroshi Kimura,et al.  Kinetics of Core Histones in Living Human Cells , 2001, The Journal of cell biology.

[41]  Tom Misteli,et al.  Spatial proximity of translocation-prone gene loci in human lymphomas , 2003, Nature Genetics.

[42]  J. Lawrence,et al.  U2 and U1 snRNA gene loci associate with coiled bodies , 1995, Journal of cellular biochemistry.

[43]  Thomas Cremer,et al.  Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells , 2004, Chromosome Research.

[44]  H. Tanke,et al.  Induction of p21 mRNA Synthesis After Short-wavelength UV Light Visualized in Individual Cells by RNA FISH , 2002, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[45]  Wendy A Bickmore,et al.  Chromatin Motion Is Constrained by Association with Nuclear Compartments in Human Cells , 2002, Current Biology.

[46]  J. Lawrence,et al.  Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains. , 2002, Journal of structural biology.

[47]  George H. Patterson,et al.  A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells , 2002, Science.

[48]  S. Kosak,et al.  The undiscovered country: chromosome territories and the organization of transcription. , 2002, Developmental cell.

[49]  Thomas Cremer,et al.  Radial chromatin positioning is shaped by local gene density, not by gene expression , 2007, Chromosoma.

[50]  A. F. Neuwald,et al.  Proteomic analysis of interchromatin granule clusters. , 2004, Molecular biology of the cell.

[51]  Joseph Rosenecker,et al.  Transcription-dependent spatial arrangements of CFTR and adjacent genes in human cell nuclei , 2004, The Journal of cell biology.

[52]  J. Mcneil,et al.  Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains , 2003, The Journal of cell biology.

[53]  H. Tanke,et al.  A glue for heterochromatin maintenance , 2005, The Journal of cell biology.