Long-Range Interactions between Three Transcriptional Enhancers, Active Vκ Gene Promoters, and a 3′ Boundary Sequence Spanning 46 Kilobases

ABSTRACT The mouse immunoglobulin kappa (Igκ) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igκ alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vκ gene promoters. In addition, the 5′ region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3′ with 3′ boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the β-globin locus and suggest a dynamic modulation of the spatial organization of the active Igκ locus. Chromatin immunoprecipitation experiments reveal that the interacting Igκ gene cis-acting sequences are associated with AP-4, E47, and p65NF-κB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-κB still exhibit mutual interactions between the Igκ gene enhancers.

[1]  Wouter de Laat,et al.  The β-globin nuclear compartment in development and erythroid differentiation , 2003, Nature Genetics.

[2]  Ulrich Siebenlist,et al.  Requirement for NF-κB in osteoclast and B-cell development , 1997 .

[3]  J. Seidman,et al.  A mutant immunoglobulin light chain is formed by aberrant DNA- and RNA-splicing events , 1980, Nature.

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

[5]  J. Strouboulis,et al.  Multiple interactions between regulatory regions are required to stabilize an active chromatin hub. , 2004, Genes & development.

[6]  F. Alt,et al.  Developmentally controlled and tissue-specific expression of unrearranged VH gene segments , 1985, Cell.

[7]  R. Perry,et al.  The role of the κ enhancer and its binding factor NF-κB in the developmental regulation of κ gene transcription , 1987, Cell.

[8]  D. Agard,et al.  Specific interactions of chromatin with the nuclear envelope: positional determination within the nucleus in Drosophila melanogaster. , 1996, Molecular biology of the cell.

[9]  D. Gray,et al.  Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes , 2001, Nature Immunology.

[10]  A. Fisher,et al.  Nuclear organisation and gene expression. , 2002, Current opinion in cell biology.

[11]  P. Cockerill,et al.  Transcription termination and chromatin structure of the active immunoglobulin kappa gene locus. , 1986, The Journal of biological chemistry.

[12]  U. Schibler,et al.  The synthesis and processing of the messenger RNAs specifying heavy and light chain immunoglobulins in MPC-11 cells , 1978, Cell.

[13]  A. Murray,et al.  Interphase chromosomes undergo constrained diffusional motion in living cells , 1997, Current Biology.

[14]  T Leanderson,et al.  Transcription factor AP-4 is a ligand for immunoglobulin-kappa promoter E-box elements. , 2001, The Biochemical journal.

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

[16]  B. V. Van Ness,et al.  Developmental Regulation of the κ Locus Involves Both Positive and Negative Sequence Elements in the 3′ Enhancer That Affect Synergy with the Intron Enhancer* , 1999, The Journal of Biological Chemistry.

[17]  E. Coligan.  Current protocols in immunology , 1991 .

[18]  J. Dekker,et al.  Capturing Chromosome Conformation , 2002, Science.

[19]  Arun J Sharma,et al.  p300 Mediates Transcriptional Stimulation by the Basic Helix-Loop-Helix Activators of the Insulin Gene , 1998, Molecular and Cellular Biology.

[20]  M. Kuehl,et al.  RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene. , 1980, Nature.

[21]  Frank Grosveld,et al.  Spatial organization of gene expression: the active chromatin hub , 2003, Chromosome Research.

[22]  J. Seidman,et al.  Two kappa immunoglobulin genes are expressed in the myeloma S107 , 1981, Cell.

[23]  M. Neuberger,et al.  The immunoglobulin kappa locus contains a second, stronger B‐cell‐specific enhancer which is located downstream of the constant region. , 1989, The EMBO journal.

[24]  S. Berger,et al.  A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex. , 1999, Molecular cell.

[25]  Richard A Flavell,et al.  Long-range intrachromosomal interactions in the T helper type 2 cytokine locus , 2004, Nature Immunology.

[26]  S. Kosak,et al.  A genetic analysis of chromosome territory looping: diverse roles for distal regulatory elements , 2004, Chromosome Research.

[27]  D. Spector,et al.  Nuclear organization and gene expression. , 1996, Experimental cell research.

[28]  D. Jackson,et al.  The principles of nuclear structure , 2004, Chromosome Research.

[29]  M. Gellert.  V(D)J recombination: RAG proteins, repair factors, and regulation. , 2002, Annual review of biochemistry.

[30]  S. M. Rose,et al.  Differentiation-dependent chromatin alterations precede and accompany transcription of immunoglobulin light chain genes. , 1984, The Journal of biological chemistry.

[31]  Matthias Merkenschlager,et al.  Association of Transcriptionally Silent Genes with Ikaros Complexes at Centromeric Heterochromatin , 1997, Cell.

[32]  D. Baltimore,et al.  Immunoglobulin gene transcription is activated by downstream sequence elements , 1983, Cell.

[33]  C. Murre,et al.  E protein function in lymphocyte development. , 2002, Annual review of immunology.

[34]  V. Corces,et al.  Setting the Boundaries of Chromatin Domains and Nuclear Organization , 2002, Cell.

[35]  T. Pederson.  The spatial organization of the genome in mammalian cells. , 2004, Current opinion in genetics & development.

[36]  M. Atchison,et al.  BSAP Can Repress Enhancer Activity by Targeting PU.1 Function , 2000, Molecular and Cellular Biology.

[37]  M. Nussenzweig,et al.  OcaB Is Required for Normal Transcription and V(D)J Recombination of a Subset of Immunoglobulin κ Genes , 2002, Cell.

[38]  R. Grosschedl,et al.  Matrix Attachment Region-Dependent Function of the Immunoglobulin μ Enhancer Involves Histone Acetylation at a Distance without Changes in Enhancer Occupancy , 2001, Molecular and Cellular Biology.

[39]  D. Baltimore,et al.  Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility. , 1987, Science.

[40]  J. Miller,et al.  Human erythroid porphobilinogen deaminase exists in 2 splice variants. , 2001, Blood.

[41]  P. Cockerill,et al.  Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites , 1986, Cell.

[42]  M. Roque,et al.  A developmentally modulated chromatin structure at the mouse immunoglobulin kappa 3' enhancer , 1996, Molecular and cellular biology.

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

[44]  W. Garrard,et al.  Yeast artificial chromosome contigs reveal that distal variable-region genes reside at least 3 megabases from the joining regions in the murine immunoglobulin kappa locus. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[45]  P. Cook.  The organization of replication and transcription. , 1999, Science.

[46]  G. J. Brakenhoff,et al.  Four-dimensional imaging of chromatin dynamics during the assembly of the interphase nucleus , 2004, Chromosome Research.

[47]  W. Garrard,et al.  Assembly and analysis of the mouse immunoglobulin kappa gene sequence , 2004, Immunogenetics.

[48]  C. Keller,et al.  Human Mcm proteins at a replication origin during the G1 to S phase transition. , 2002, Nucleic acids research.

[49]  J. Seidman,et al.  Nucleic acid and protein sequences of phosphocholine-binding light chains , 1981, The Journal of experimental medicine.

[50]  G. Felsenfeld,et al.  CTCF tethers an insulator to subnuclear sites, suggesting shared insulator mechanisms across species. , 2004, Molecular cell.

[51]  Wolf Reik,et al.  Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops , 2004, Nature Genetics.

[52]  J. Dekker.  A closer look at long-range chromosomal interactions. , 2003, Trends in biochemical sciences.

[53]  A. Shaffer,et al.  In Vivo Occupancy of the κ Light Chain Enhancers in Primary Pro- and Pre-B Cells: A Model for κ Locus Activation , 1997 .

[54]  W. Garrard,et al.  Chromatin Structural Analyses of the Mouse Igκ Gene Locus Reveal New Hypersensitive Sites Specifying a Transcriptional Silencer and Enhancer* , 2002, The Journal of Biological Chemistry.

[55]  S. Tonegawa,et al.  Transcription of mouse kappa chain genes: implications for allelic exclusion. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[56]  T. Yao,et al.  Regulation of E2A Activities by Histone Acetyltransferases in B Lymphocyte Development* , 2003, The Journal of Biological Chemistry.

[57]  W. Marshall,et al.  Order and Disorder in the Nucleus , 2002, Current Biology.

[58]  A. Feeney,et al.  Both E12 and E47 allow commitment to the B cell lineage. , 1997, Immunity.

[59]  W. Garrard,et al.  Immunoglobulin kappa gene enhancers synergistically activate gene expression but independently determine chromatin structure. , 1992, The Journal of biological chemistry.

[60]  W. Murphy,et al.  Structure of the 5' ends of immunoglobulin genes: a novel conserved sequence. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[61]  Erik Splinter,et al.  Looping and interaction between hypersensitive sites in the active beta-globin locus. , 2002, Molecular cell.

[62]  W. Garrard,et al.  A targeted kappa immunoglobulin gene containing a deletion of the nuclear matrix association region exhibits spontaneous hyper-recombination in pre-B cells. , 1998, Molecular immunology.

[63]  Tom Misteli,et al.  Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins , 2004, Molecular and Cellular Biology.

[64]  Cameron S. Osborne,et al.  Long-range chromatin regulatory interactions in vivo , 2002, Nature Genetics.

[65]  W. Bickmore,et al.  Considering Nuclear Compartmentalization in the Light of Nuclear Dynamics , 2003, Cell.

[66]  D. Livingston,et al.  Interaction and functional collaboration of p300/CBP and bHLH proteins in muscle and B-cell differentiation. , 1996, Genes & development.

[67]  Roy Riblet,et al.  Subnuclear Compartmentalization of Immunoglobulin Loci During Lymphocyte Development , 2002, Science.

[68]  Ruth R. E. Williams.  Transcription and the territory: the ins and outs of gene positioning. , 2003, Trends in genetics : TIG.

[69]  M. Atchison,et al.  Functional characterization of the developmentally controlled immunoglobulin kappa 3' enhancer: regulation by Id, a repressor of helix-loop-helix transcription factors , 1991, Molecular and cellular biology.