臺灣博碩士論文加值系統

NF-E2 是人類紅血球蛋白質基因的轉錄活化因子，主要是調
控一些人類紅血球及巨核細胞譜系中的特定基因。在本論文中我證明
了哺乳動物 NF-E2 的大次單元體 p45 在細胞中會被 sumoylation。
藉由在試管中所進行的 sumoylation 反應以及細胞中 DNA 轉染實
驗，我觀察到 sumoylation 是作用於人類 NF-E2 大次單元體 p45 的
Lysine 368 (K368) 的位置上。
我更進一步發現 sumolyation 的 p45 會造成 NF-E2 對
DNA 結合能力的增加，而促進 NF-E2 對人類紅血球蛋白質基因的轉
譯活化的能力。更有趣的是我發現在表現血紅素蛋白的人類紅血球
K562 細胞株中, 被 sumoylation 的 p45/NF-E2 核體是位於真染色
質 (euchromatin) 的位置中，且與 beta-血紅素基因、promyelocytic
leukemia protein (PML) POD 核體 (nuclear bodies)、SUMO 核體、
RNA 聚合酶 (RNA polymerase II) 在一起。
而在細胞內的蛋白質與基因結合情況呢？我藉由核染色質
(Chromatin) 免疫沉澱分析技術 (ChIP) 在永久性轉染 (stable
transfection) 人類 NF-E2 大次單元體 p45 的人類紅血球 K562 細胞
株中，觀察到具有完整sumoylation 位置的人類 NF-E2 大次單元體
p45 才具有結合至 β-globin-locus-control-region (β-LCR) 之 DNaseI
高敏感區 (HS) 的功能。
最後我利用永久性轉染 (stable transfection assay) 來證明在
小鼠 NF-E2 大次單元體 p45 缺乏的血球細胞株 CB3 中，只有具有
10
完整的 sumoylation 位置的人類 NF-E2 大次單元體 p45，才擁有回
復 β-血紅蛋白表現的能力。而突變的人類 NF-E2 大次單元體 p45
(K368R) 並不具有這樣的能力。
綜合上述數據，我提出了哺乳動物 β-血紅蛋白基因會被
sumoylated 哺乳動物 NF-E2 大次單元體 p45 活化的模式。

NF-E2 is a transcription activator for the regulation of a number of
erythroid- and megakaryocytic lineage-specific genes. Here I present
evidence that the large subunit of mammalian NF-E2, p45, is sumoylated
in vivo, in human erythroid K562 cells and in mouse fetal liver. By in
vitro sumoylation reaction and DNA transfection experiments, I show that
the sumoylation occurs at lysine 368 (K368) of human p45/NF-E2.
Furthermore, p45 sumoylation enhances the transactivation capability of
NF-E2, and this is accompanied by an increase of the NF-E2
DNA-binding affinity. More interestingly, I have found that in K562 cells,
the β-globin gene loci in the euchromatin regions are predominantly
colocalized with the nuclear bodies promyelocytic leukemia protein
(PML) oncogenic domains (PODs) that are enriched with the PML,
SUMO-1, RNA polymerase II and sumoylatable p45/NF-E2. Chromatin
immunoprecipitation assay (ChIP) further showed that intact sumoylation
site of p45/NF-E2 is required for its binding to the DNase I
hypersensitive sites (HS) of the β�n-globin locus-control-region (β-LCR).
Finally, I demonstrated by stable transfection assay that only the wild
type p45, but not its mutant form p45 (K368R), could efficiently rescue
the β-�nglobin gene expression in the p45-null, erythroid cell line CB3.
These data together point to a model of mammalian β-like globin
gene activation by sumoylated p45/NF-E2 in erythroid cells.

國立陽明大學博士班研究生論文審定證明書........................................2
國立陽明大學學位論文電子檔著作權授權書........................................3
Acknowledgements.....................................................................................4
Table of content...........................................................................................6
Abstract in Chinese (中文摘要) .................................................................9
Abstract .....................................................................................................10
Abstract .....................................................................................................11
Introduction...............................................................................................12
Globin........................................................................................................12
NF-E2........................................................................................................13
Nuclear Bodies (NB) ................................................................................15
SUMO (small ubiquitin-like modifier).....................................................16
Specific Aims ............................................................................................17
Materials and Methods..............................................................................19
Cell Culture...............................................................................................19
Plasmids and Antibodies...........................................................................19
Protein Expression. ...................................................................................20
In Vitro Sumoylation and Desumoylation Assay. .....................................20
DNA Transfection and Luciferase Assay..................................................21
Purification of His-Tagged proteins from Cell Extracts...........................21
Preparation of Nuclear Extract. ................................................................21
Ammonium sulfate precipitation. .............................................................22
Analysis of p45 sumoylation in mouse fetal liver. ...................................23
Immunoprecipitation and Western blotting.. ............................................23
7
Electrophoretic Mobility Shift Assay (EMSA). .......................................23
Immunostaining. .......................................................................................24
DNA Fluorescence In Situ Hybridization (DNA-FISH). .........................24
G-Banding Chromosome Analysis. ..........................................................25
Immuno-DNA-FISH.................................................................................26
Image Analysis..........................................................................................26
Chromatin Immunoprecipitation (ChIP)...................................................26
Northern Blot Analysis. ............................................................................27
Results.......................................................................................................27
Sumoylation of p45 In Vitro......................................................................27
Sumoylation of p45 In Vivo. .....................................................................28
Biological Effects of p45 Sumoylation.....................................................30
Transactivation. ...........................................................................30
DNA binding affinity...................................................................31
Erythroid Cell-Specific Association of Human β-Globin Locus with PML
Nuclear Bodies (POD) Containing Sumoylated p45/NF-E2 and RNA
Polymerase II. ...........................................................................................32
Co-staining of anti-p45, anti-SUMO-1 and anti-PML................32
Immuno-DNA-FISH of the human β-.........................................34
Binding In Vivo of the Wild Type p45, But Not p45 (K368R), to the
β-LCR in K562 Cells. ...............................................................................36
Rescue of β-Globin Gene Expression in CB3 Cells by Wild Type p45, But
Not by p45 (K368R). ................................................................................36
Discussion .................................................................................................37
References.................................................................................................42
8
Figure 1. 1 The human globin genes cluster..........................................51
Figure 1. 2 Schematic representation of the domain organization of p45
...................................................................................................................53
Figure 1. 3 Transcription factories in POD............................................54
Figure 1. 4 Sumoylation.........................................................................54
Figure 1. 5 Phosphorylation of p45 .......................................................55
Figure 1. Sumoylation of p45/NF-E2 in vitro...........................................56
Figure 2. Sumoylation of p45/NF-E2 in vivo. ..........................................60
Figure 3. Enhancement of transactivation capability of p45/NF-E2 by
sumoylation...............................................................................................63
Figure 4. Effects of p45 sumoylation on NF-E2/DNA binding................69
Figure 5. Immunofluoresence staining of K562 cells...............................71
Figure 6. G-banding karyotype and DNA-FISH ......................................72
Figure 7. Immuno-DNA-FISH. ................................................................73
Figure 8.Chromatin immunoprecipitation assay (ChIP)...........................74
Figure 9. Rescue of adult globin gene expression in CB3 cells. ..............76
Figure 10. Model of transcriptional activation of human β-like globin
genes by p45/NF-E2 sumoylation. ...........................................................78

Amrolia, P.J., Ramamurthy, L., Saluja, D., Tanese, N., Jane, S.M. and
Cunningham, J.M. (1997) The activation domain of the enhancer
binding protein p45NF-E2 interacts with TAFII130 and mediates
long-range activation of the alpha- and beta-globin gene loci in an
erythroid cell line. Proc Natl Acad Sci U S A, 94, 10051-10056.
Andrews, N.C. (1998) The NF-E2 transcription factor. Int J Biochem Cell
Biol, 30, 429-432.
Andrews, N.C., Erdjument-Bromage, H., Davidson, M.B., Tempst, P. and
Orkin, S.H. (1993) Erythroid transcription factor NF-E2 is a
haematopoietic-specific basic-leucine zipper protein. Nature, 362,
722-728.
Andrews, N.C., Kotkow, K.J., Ney, P.A., Erdjument-Bromage, H.,
Tempst, P. and Orkin, S.H. (1993) The ubiquitous subunit of
erythroid transcription factor NF-E2 is a small basic-leucine zipper
protein related to the v-maf oncogene. Proc Natl Acad Sci U S A,
90, 11488-11492.
Armstrong, J.A. and Emerson, B.M. (1996) NF-E2 disrupts chromatin
structure at human beta-globin locus control region hypersensitive
site 2 in vitro. Mol Cell Biol, 16, 5634-5644.
Bean, T.L. and Ney, P.A. (1997) Multiple regions of p45 NF-E2 are
required for beta-globin gene expression in erythroid cells. Nucleic
Acids Res, 25, 2509-2515.
Bernardi, R., Scaglioni, P.P., Bergmann, S., Horn, H.F., Vousden, K.H.
and Pandolfi, P.P. (2004) PML regulates p53 stability by
sequestering Mdm2 to the nucleolus. Nat Cell Biol, 6, 665-672.
Blank, V., Kim, M.J. and Andrews, N.C. (1997) Human MafG is a
functional partner for p45 NF-E2 in activating globin gene
43
expression. Blood, 89, 3925-3935.
Bulger, M. and Groudine, M. (1999) Looping versus linking: toward a
model for long-distance gene activation. Genes Dev, 13,
2465-2477.
Casteel, D., Suhasini, M., Gudi, T., Naima, R. and Pilz, R.B. (1998)
Regulation of the erythroid transcription factor NF-E2 by cyclic
adenosine monophosphate-dependent protein kinase. Blood, 91,
3193-3201.
Caterina, J.J., Donze, D., Sun, C.W., Ciavatta, D.J. and Townes, T.M.
(1994) Cloning and functional characterization of LCR-F1: a bZIP
transcription factor that activates erythroid-specific, human globin
gene expression. Nucleic Acids Res, 22, 2383-2391.
Chan, J.Y., Han, X.L. and Kan, Y.W. (1993) Isolation of cDNA encoding
the human NF-E2 protein. Proc Natl Acad Sci U S A, 90,
11366-11370.
Chang, K.S., Fan, Y.H., Andreeff, M., Liu, J. and Mu, Z.M. (1995) The
PML gene encodes a phosphoprotein associated with the nuclear
matrix. Blood, 85, 3646-3653.
Chen, W.Y., Lee, W.C., Hsu, N.C., Huang, F. and Chung, B.C. (2004)
SUMO modification of repression domains modulates function of
nuclear receptor 5A1 (steroidogenic factor-1). J Biol Chem, 279,
38730-38735.
Chen, X., Wen, S., Fukuda, M.N., Gavva, N.R., Hsu, D., Akama, T.O.,
Yang-Feng, T. and Shen, C.-K.J. (2001) Human ITCH is a
coregulator of the hematopoietic transcription factor NF-E2.
Genomics, 73, 238-241.
Cheng, X., Reginato, M.J., Andrews, N.C. and Lazar, M.A. (1997) The
transcriptional integrator CREB-binding protein mediates positive
cross talk between nuclear hormone receptors and the
44
hematopoietic bZip protein p45/NF-E2. Mol Cell Biol, 17,
1407-1416.
Cox, T.C., Bawden, M.J., Martin, A. and May, B.K. (1991) Human
erythroid 5-aminolevulinate synthase: promoter analysis and
identification of an iron-responsive element in the mRNA. Embo J,
10, 1891-1902.
Cremer, T. and Cremer, C. (2001) Chromosome territories, nuclear
architecture and gene regulation in mammalian cells. Nat Rev
Genet, 2, 292-301.
Daftari, P., Gavva, N.R. and Shen, C.-K.J. (1999) Distinction between
AP1 and NF-E2 factor-binding at specific chromatin regions in
mammalian cells. Oncogene, 18, 5482-5486.
de Jong, L., Grande, M.A., Mattern, K.A., Schul, W. and van Driel, R.
(1996) Nuclear domains involved in RNA synthesis, RNA
processing, and replication. Crit Rev Eukaryot Gene Expr, 6,
215-246.
de Laat, W. and Grosveld, F. (2003) Spatial organization of gene
expression: the active chromatin hub. Chromosome Res, 11,
447-459.
Engel, J.D. and Tanimoto, K. (2000) Looping, linking, and chromatin
activity: new insights into beta-globin locus regulation. Cell, 100,
499-502.
Eskiw, C.H., Dellaire, G., Mymryk, J.S. and Bazett-Jones, D.P. (2003)
Size, position and dynamic behavior of PML nuclear bodies
following cell stress as a paradigm for supramolecular trafficking
and assembly. J Cell Sci, 116, 4455-4466.
Francastel, C., Magis, W. and Groudine, M. (2001) Nuclear relocation of
a transactivator subunit precedes target gene activation. Proc Natl
Acad SciU S A, 98, 12120-12125.
45
Fu, X.D. and Maniatis, T. (1990) Factor required for mammalian
spliceosome assembly is localized to discrete regions in the nucleus.
Nature, 343, 437-441.
Gall, J.G. (2000) Cajal bodies: the first 100 years. Annu Rev Cell Dev
Biol, 16, 273-300.
Gong, Q.H., McDowell, J.C. and Dean, A. (1996) Essential role of NF-E2
in remodeling of chromatin structure and transcriptional activation
of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the
beta-globin locus control region. Mol Cell Biol, 16, 6055-6064.
Goodson, M.L., Hong, Y., Rogers, R., Matunis, M.J., Park-Sarge, O.K.
and Sarge, K.D. (2001) Sumo-1 modification regulates the DNA
binding activity of heat shock transcription factor 2, a
promyelocytic leukemia nuclear body associated transcription
factor. J Biol Chem, 276, 18513-18518.
Grande, M.A., van der Kraan, I., de Jong, L. and van Driel, R. (1997)
Nuclear distribution of transcription factors in relation to sites of
transcription and RNA polymerase II. J Cell Sci, 110 (Pt 15),
1781-1791.
Hakli, M., Karvonen, U., Janne, O.A. and Palvimo, J.J. (2005) SUMO-1
promotes association of SNURF (RNF4) with PML nuclear bodies.
Exp Cell Res, 304, 224-233.
Herrmann, A., Sommer, U., Pranada, A.L., Giese, B., Kuster, A., Haan,
S., Becker, W., Heinrich, P.C. and Muller-Newen, G. (2004)
STAT3 is enriched in nuclear bodies. J Cell Sci, 117, 339-349.
Hong, Y., Rogers, R., Matunis, M.J., Mayhew, C.N., Goodson, M.L.,
Park-Sarge, O.K., Sarge, K.D. and Goodson, M. (2001) Regulation
of heat shock transcription factor 1 by stress-induced SUMO-1
modification. J Biol Chem, 276, 40263-40267.
Hung, H.L., Kim, A.Y., Hong, W., Rakowski, C. and Blobel, G.A. (2001)
46
Stimulation of NF-E2 DNA binding by CREB-binding protein
(CBP)-mediated acetylation. J Biol Chem, 276, 10715-10721.
Igarashi, K., Itoh, K., Motohashi, H., Hayashi, N., Matuzaki, Y.,
Nakauchi, H., Nishizawa, M. and Yamamoto, M. (1995) Activity
and expression of murine small Maf family protein MafK. J Biol
Chem, 270, 7615-7624.
Igarashi, K., Kataoka, K., Itoh, K., Hayashi, N., Nishizawa, M. and
Yamamoto, M. (1994) Regulation of transcription by dimerization
of erythroid factor NF-E2 p45 with small Maf proteins. Nature,
367, 568-572.
Johnson, E.S. (2004) Protein modification by SUMO. Annu Rev Biochem,
73, 355-382.
Johnson, K.D., Christensen, H.M., Zhao, B. and Bresnick, E.H. (2001)
Distinct mechanisms control RNA polymerase II recruitment to a
tissue-specific locus control region and a downstream promoter.
Mol Cell, 8, 465-471.
Kiesslich, A., von Mikecz, A. and Hemmerich, P. (2002) Cell
cycle-dependent association of PML bodies with sites of active
transcription in nuclei of mammalian cells. J Struct Biol, 140,
167-179.
Kotkow, K.J. and Orkin, S.H. (1995) Dependence of globin gene
expression in mouse erythroleukemia cells on the NF-E2
heterodimer. Mol Cell Biol, 15, 4640-4647.
Lamond, A.I. and Earnshaw, W.C. (1998) Structure and function in the
nucleus. Science, 280, 547-553.
Lamond, A.I. and Sleeman, J.E. (2003) Nuclear substructure and
dynamics. Curr Biol, 13, R825-828.
LaMorte, V.J., Dyck, J.A., Ochs, R.L. and Evans, R.M. (1998)
Localization of nascent RNA and CREB binding protein with the
47
PML-containing nuclear body. Proc Natl Acad Sci U S A, 95,
4991-4996.
Li, Q., Peterson, K.R., Fang, X. and Stamatoyannopoulos, G. (2002)
Locus control regions. Blood, 100, 3077-3086.
Li, Y.C., Lee, C., Sanoudou, D., Hseu, T.H., Li, S.Y., Lin, C.C. and Hsu,
T.H. (2000) Interstitial colocalization of two cervid satellite DNAs
involved in the genesis of the Indian muntjac karyotype.
Chromosome Res, 8, 363-373.
Liu, J.J., Hou, S.C. and Shen, C.-K.J. (2003) Erythroid gene suppression
by NF-kappa B. J Biol Chem, 278, 19534-19540.
Liu, Q. and Dreyfuss, G. (1996) A novel nuclear structure containing the
survival of motor neurons protein. Embo J, 15, 3555-3565.
Lu, S.J., Rowan, S., Bani, M.R. and Ben-David, Y. (1994) Retroviral
integration within the Fli-2 locus results in inactivation of the
erythroid transcription factor NF-E2 in Friend erythroleukemias:
evidence that NF-E2 is essential for globin expression. Proc Natl
Acad Sci U S A, 91, 8398-8402.
Matera, A.G. (1999) Nuclear bodies: multifaceted subdomains of the
interchromatin space. Trends Cell Biol, 9, 302-309.
Mignotte, V., Eleouet, J.F., Raich, N. and Romeo, P.H. (1989) Cis- and
trans-acting elements involved in the regulation of the erythroid
promoter of the human porphobilinogen deaminase gene. Proc Natl
Acad Sci U S A, 86, 6548-6552.
Misteli, T. and Spector, D.L. (1998) The cellular organization of gene
expression. Curr Opin Cell Biol, 10, 323-331.
Moi, P. and Kan, Y.W. (1990) Synergistic enhancement of globin gene
expression by activator protein-1-like proteins. Proc Natl Acad Sci
U S A, 87, 9000-9004.
Muller, S., Miller, W.H., Jr. and Dejean, A. (1998) Trivalent antimonials
48
induce degradation of the PML-RAR oncoprotein and
reorganization of the promyelocytic leukemia nuclear bodies in
acute promyelocytic leukemia NB4 cells. Blood, 92, 4308-4316.
Naumann, S., Reutzel, D., Speicher, M. and Decker, H.J. (2001)
Complete karyotype characterization of the K562 cell line by
combined application of G-banding, multiplex-fluorescence in situ
hybridization, fluorescence in situ hybridization, and comparative
genomic hybridization. Leuk Res, 25, 313-322.
Negorev, D. and Maul, G.G. (2001) Cellular proteins localized at and
interacting within ND10/PML nuclear bodies/PODs suggest
functions of a nuclear depot. Oncogene, 20, 7234-7242.
Ney, P.A., Sorrentino, B.P., McDonagh, K.T. and Nienhuis, A.W. (1990)
Tandem AP-1-binding sites within the human beta-globin
dominant control region function as an inducible enhancer in
erythroid cells. Genes Dev, 4, 993-1006.
Nudel, U., Salmon, J.E., Terada, M., Bank, A., Rifkind, R.A. and Marks,
P.A. (1977) Differential effects of chemical inducers on expression
of beta globin genes in murine erythroleukemia cells. Proc Natl
Acad Sci U S A, 74, 1100-1104.
Osborne, C.S., Chakalova, L., Brown, K.E., Carter, D., Horton, A.,
Debrand, E., Goyenechea, B., Mitchell, J.A., Lopes, S., Reik, W.
and Fraser, P. (2004) Active genes dynamically colocalize to
shared sites of ongoing transcription. Nat Genet, 36, 1065-1071.
Regad, T. and Chelbi-Alix, M.K. (2001) Role and fate of PML nuclear
bodies in response to interferon and viral infections. Oncogene, 20,
7274-7286.
Salomoni, P. and Pandolfi, P.P. (2002) The role of PML in tumor
suppression. Cell, 108, 165-170.
Sawado, T., Halow, J., Bender, M.A. and Groudine, M. (2003) The beta
49
-globin locus control region (LCR) functions primarily by
enhancing the transition from transcription initiation to elongation.
Genes Dev, 17, 1009-1018.
Sawado, T., Igarashi, K. and Groudine, M. (2001) Activation of
beta-major globin gene transcription is associated with recruitment
of NF-E2 to the beta-globin LCR and gene promoter. Proc Natl
Acad Sci U S A, 98, 10226-10231.
Schul, W., van Driel, R. and de Jong, L. (1998) Coiled bodies and U2
snRNA genes adjacent to coiled bodies are enriched in factors
required for snRNA transcription. Mol Biol Cell, 9, 1025-1036.
Shiels, C., Islam, S.A., Vatcheva, R., Sasieni, P., Sternberg, M.J.,
Freemont, P.S. and Sheer, D. (2001) PML bodies associate
specifically with the MHC gene cluster in interphase nuclei. J Cell
Sci, 114, 3705-3716.
Shivdasani, R.A. and Orkin, S.H. (1995) Erythropoiesis and globin gene
expression in mice lacking the transcription factor NF-E2. Proc
Natl Acad Sci U S A, 92, 8690-8694.
Shivdasani, R.A., Rosenblatt, M.F., Zucker-Franklin, D., Jackson, C.W.,
Hunt, P., Saris, C.J. and Orkin, S.H. (1995) Transcription factor
NF-E2 is required for platelet formation independent of the actions
of thrombopoietin/MGDF in megakaryocyte development. Cell, 81,
695-704.
Spector, D.L. (1993) Macromolecular domains within the cell nucleus.
Annu Rev Cell Biol, 9, 265-315.
Sun, Y., Durrin, L.K. and Krontiris, T.G. (2003) Specific interaction of
PML bodies with the TP53 locus in Jurkat interphase nuclei.
Genomics, 82, 250-252.
Takahashi, Y., Lallemand-Breitenbach, V., Zhu, J. and de The, H. (2004)
PML nuclear bodies and apoptosis. Oncogene, 23, 2819-2824.
50
Talbot, D. and Grosveld, F. (1991) The 5'HS2 of the globin locus control
region enhances transcription through the interaction of a
multimeric complex binding at two functionally distinct NF-E2
binding sites. Embo J, 10, 1391-1398.
Verschure, P.J., van Der Kraan, I., Manders, E.M. and van Driel, R.
(1999) Spatial relationship between transcription sites and
chromosome territories. J Cell Biol, 147, 13-24.
von Mikecz, A., Zhang, S., Montminy, M., Tan, E.M. and Hemmerich, P.
(2000) CREB-binding protein (CBP)/p300 and RNA polymerase II
colocalize in transcriptionally active domains in the nucleus. J Cell
Biol, 150, 265-273.
Wang, I.F., Reddy, N.M. and Shen, C.-K.J. (2002) Higher order
arrangement of the eukaryotic nuclear bodies. Proc Natl Acad Sci
U S A, 99, 13583-13588.
Wang, J., Feng, X.H. and Schwartz, R.J. (2004) SUMO-1 modification
activated GATA4 dependent cardiogenic gene activity. J Biol
Chem.
Wang, J., Shiels, C., Sasieni, P., Wu, P.J., Islam, S.A., Freemont, P.S. and
Sheer, D. (2004) Promyelocytic leukemia nuclear bodies associate
with transcriptionally active genomic regions. J Cell Biol, 164,
515-526.
Wen, S.C., Roder, K., Hu, K.Y., Rombel, I., Gavva, N.R., Daftari, P.,
Kuo, Y.Y., Wang, C. and Shen, C.-K.J. (2000) Loading of
DNA-binding factors to an erythroid enhancer. Mol Cell Biol, 20,
1993-2003.
Xing, Y., Johnson, C.V., Dobner, P.R. and Lawrence, J.B. (1993) Higher
level organization of individual gene transcription and RNA
splicing. Science, 259, 1326-1330.
Zeng, C., Kim, E., Warren, S.L. and Berget, S.M. (1997) Dynamic
51
relocation of transcription and splicing factors dependent upon
transcriptional activity. Embo J, 16, 1401-1412.
Zhang, G., Taneja, K.L., Singer, R.H. and Green, M.R. (1994)
Localization of pre-mRNA splicing in mammalian nuclei. Nature,
372, 809-812.