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First published online 16 November 2004
doi: 10.1242/jcs.01537

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Dynamic relocation of epigenetic chromatin markers reveals an active role of constitutive heterochromatin in the transition from proliferation to quiescence

Sergei A. Grigoryev^1,*, Tatiana Nikitina^2,*, John R. Pehrson³, Prim B. Singh⁴ and Christopher L. Woodcock^2,

¹ Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, PA 17033, USA
² Biology Department, University of Massachusetts, Amherst, MA 01003, USA
³ University of Pennsylvania, Department of Animal Biology, Philadelphia, PA 19104-6049, USA
⁴ Nuclear Reprogramming Laboratory, Division of Gene Expression and Development, The Roslin Institute (Edinburgh), Midlothian, EH25 9PS, UK

View larger version (58K): [in a new window]   Fig. 1. macroH2A in quiescent mouse lymphocytes and hepatocytes. Quiescent lymphocytes from mouse spleen (a-e) and liver cells (f-h) stained with antibodies against macroH2A (a,f) and Hoechst 22358 (b,g). Double-staining with antibodies against macroH2A and Hoechst (c,h), macroH2A and CENPB (d) and CENPB and Hoechst (e).

View larger version (72K): [in a new window]   Fig. 5. MacroH2A in reactivated lymphocytes. (a) Con A-activated lymphocytes from mouse spleen double-stained with antibodies against macroH2A and BrdU. (b) Nuclear proteins from resting (Con A -) and activated (Con A +) lymphocytes were stained with Coomassie (lanes 1, 2) and probed with antibodies against macroH2A (lanes 3, 4). (c) Fraction of proliferating, BrdU-positive lymphocyte nuclei (open columns) and fraction of nuclei with pericentromeric macroH2A distribution (solid columns), with standard errors. Lymphocytes were reactivated with Con A for 36 hours, pulsed with BrdU for 5 minutes and, at the times indicated, stained for macroH2A and BrdU. (d) Fraction of nuclei with pericentromeric macroH2A distribution. Lymphocytes were reactivated with Con A for 36 hours, then pulsed with BrdU for 1 hour [trichostatin A (TSA)] or 5 minutes (Na-but) and were incubated for 48 hours without histone deacetylase (HDAC) inhibitors (48), for the last 4 hours with 5 ng/ml TSA (TSA), or for last 24 hours with 2.5 mM Na-butyrate (Na-but). Cells were stained for macroH2A, and quantitated to reveal the distribution of macroH2A.

View larger version (48K): [in a new window]   Fig. 2. Localization of histone modifications in quiescent lymphocytes and 3T3 fibroblasts. Quiescent lymphocytes from mouse spleen (a-g) and 3T3 cells (h-m) stained with antibodies against H4K12Ac, H3K9Me2, H3K9Me3, and Hoechst as indicated. Overlays double-stained with antibodies against H4K12Ac and Hoechst (c), H3K9Me2 and Hoechst (d,j) and H3K9Me3 and Hoechst (m). The positions of line profiles shown on panels a and b are indicated by the broken lines on panels c and d, respectively. Vertical arrows on panels a and b denote the approximate locations of the apocentric zone where H4K12Ac is absent, and H3K9Me2 maximal.

View larger version (57K): [in a new window]   Fig. 3. Histone modifications in reactivated lymphocytes. (a,b) Nuclear proteins from resting (Con A -) and Con A-activated (Con A +) spleen lymphocytes were separated by SDS-PAGE and stained (lanes 1, 2, 5, and 6) or probed for H4AcK12 (lanes 3, 4) and H3K9Me2 (lanes 7, 8). (c,d) Con A-activated lymphocytes from mouse spleen double-stained for H4K12Ac and Hoechst (c) and H3K9Me2 and Hoechst (d).

View larger version (74K): [in a new window]   Fig. 4. Localization of heterochromatin protein 1 (HP1) and Ikaros in resting and reactivated lymphocytes. Resting spleen lymphocytes (a,e) and lymphocytes treated with Con A for 36 (i) and 72 hours (b-d,f-h,j) stained for HP1 (a,b), HP1ß (e,f), Hoechst (c,g) and double-stained for HP1 and Hoechst (d), HP1ß and Hoechst (h) and Ikaros and Hoechst (i,j).

View larger version (59K): [in a new window]   Fig. 6. Reciprocal accumulation of macroH2A and HP1 in NIH/3T3 cells. (a,b) NIH/3T3 cells transfected with expression vectors encoding express-macroH2A (panels 1-9), express-macroH2A1-140 (panels 10-11), and co-expressing macroH2A and express-H2A.l (12,13), and macroH2A and express-H2A.X (14, 15). Slides were stained with -express (1,4,7,10,13,15), -HP1 (3,6), -HP1ß (9), -macroH2A (12,14) and Hoechst 33258 (2,5,8). Cells in panels 4-15 were incubated for 48 hours with 5 mM sodium butyrate.

View larger version (32K): [in a new window]   Fig. 7. Zonal partitioning and localization changes of major heterochromatin proteins in quiescent and proliferating cells. Schematic diagram showing the levels: negligible (white boxes), intermediate (grey boxes), and high (black boxes) of selected heterochromatin proteins in the distinct zones of the resting and activated lymphocytes and quiescent and proliferating NIH/3T3 cell nucleus. Asterisks indicate the changes associated with the transition from quiescence to proliferation.

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