QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 August 2004
doi: 10.1242/jcs.01328

This Article Summary Full Text Full Text (PDF) Supplemental Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sarmento, O. F. Articles by Coonrod, S. A. Search for Related Content PubMed PubMed Citation Articles by Sarmento, O. F. Articles by Coonrod, S. A.

Dynamic alterations of specific histone modifications during early murine development

¹ Department of Cell Biology, University of Virginia Health Science Center, PO Box 800732, Charlottesville, VA 22908, USA
² Laboratory of Chromatin Biology, The Rockefeller University, 230 York Avenue, New York, NY 10021, USA
³ Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA

View larger version (63K): [in a new window]   Fig. 1. Methylated lysine 9 on histone H3. (A-L) Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the Me(Lys9)H3 modification (E-H, red) and DNA (I-L, green). Chromatin staining for this modification is present with little apparent change in fluorescence intensity at all stages of pre-implantation development tested (E-H). (A,E,I) In immature germinal vesicle (GV) stage oocytes, the Me(Lys9)H3 modification co-localizes with DNA staining that surrounds the nucleolus (GV Stage SN). (B,F,J) In fertilized eggs, the maternal metaphase II plate (MII plate, asterisk) stains strongly for this modification whereas the decondensing sperm nucleus (SpN) does not stain. (C,G,K) At the pronuclear stage, the male pronucleus (MPN) also does not stain for Me(Lys9)H3, whereas the female pronucleus (FPN) stains strongly. The polar body can be seen at this pronuclear stage (PB). (D,H,L) At the blastocyst stage of development, note that chromatin from both metaphase (Met) and interphase (Int) blastomeres stain strongly for the Me(Lys9)H3 mark.

View larger version (70K): [in a new window]   Fig. 2. Methylated lysine 4 on histone H3. (A-L) Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the Me(Lys4)H3 modification (E-H, red) and DNA (I-L, green). Chromatin staining is present at all stages of pre-implantation development with little apparent change in fluorescence intensity between the stages tested (E-H). (A,E,I) In immature oocytes, the Me(Lys 4)H3 modification co-localizes with DNA staining surrounding the nucleolus. (B,F,J) In fertilized eggs, the maternal metaphase II plate (F, asterisk) stains strongly for this modification whereas the decondensing sperm nucleus does not stain (F, arrow). (C,G,K) As with the Me(Lys9)H3 mark, there was little to no staining for the Me(Lys4)H3 modification in the male pronucleus.

View larger version (57K): [in a new window]   Fig. 3. Phosphorylated serine 1 on histone H4/H2A. (A-L) Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the Ph(Ser1)H4/H2A modification (E-H, red) and nucleic acid (I-L, green). Chromatin staining is present at all stages of pre-attachment development. (B,F,J) In fertilized eggs, both the metaphase II plate (F, asterisk) and decondensing sperm nuclei (F, arrow) stain positive for this modification. (D,H,L) In the blastocyst note that levels of this modification in inner cell mass blastomeres (identified by arrow in D) appears to be lower than those seen in blastomeres of the trophectoderm.

View larger version (65K): [in a new window]   Fig. 4. Hyperacetylated histone H4. Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the hyperacetylated H4 modification (F-J, red) and nucleic acid (K-O, green). (A,F,K) In immature oocytes, hyperacetylated H4 staining is limited to condensing chromatin surrounding the nucleolus. Cumulus cells are also strongly stained by this antibody (data not shown). (B,G,L) In fertilized eggs, staining for this modification is seen in decondensing sperm nuclei (G, arrows) and is only weakly associated with the metaphase plate of maternal chromatin (G, asterisk). (D,I,N) At the four-cell stage almost no staining is observed in metaphase stage blastomeres (arrows) whereas strong staining is seen in interphase stage blastomeres. (E,J,O) However, by the blastocyst stage of development, hyperacetylated H4 staining again becomes weakly associated with metaphase chromatin (arrows). Fluorescent images represent single confocal sections, therefore only nuclei found within the optical section can be visualized.

View larger version (59K): [in a new window]   Fig. 5. Methylated arginine 17 on histone H3. Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the Me(Arg17)H3 modification (F-J, red) and nucleic acid (K-O, green). (A,F,K) Immature oocyte nuclei displayed high levels of this modification in a punctate manner throughout the entire nuclear region excluding the nucleolus. (B,G,L) At fertilization, diffuse punctate cytoplasmic Me(Arg17)H3 staining is seen with no apparent staining observed on the metaphase plate (asterisks). (C,H,M) At the pronuclear stage, however, the modification is associated with chromatin in both pronuclei at similar levels. In all of the subsequent developmental stages tested, punctate cytoplasmic staining with little or no chromatin staining is observed in metaphase stage blastomeres (J, O, arrows). There is also little or no staining in anaphase stage blastomeres (I, N, arrows). This modification also stains cumulus cell nuclei and only stains polar bodies faintly (data not shown). Fluorescent images represent single confocal sections, therefore only nuclei found within the optical section can be visualized.

View larger version (64K): [in a new window]   Fig. 6. Methylated arginine 3 on histone H4. Light and fluorescent micrographs of mouse immature oocytes, fertilized eggs and early embryonic stages stained for the Me(Arg3)H4 modification (B,E,I,L,O,R, red) and nucleic acid (C,F,J,M,P,S, green). (A-C) This modification is also found in a punctate manner throughout the nucleus of the immature oocyte. (D-F) At fertilization, this modification appears to be particularly affected by the egg cytoplasm, as it is undetectable in both the egg cytoplasm and the metaphase plate chromatin of fertilized oocytes (asterisks). (H-J) At the pronuclear stage, nuclear Me(Arg3)H4 staining becomes apparent in some zygotes (data not shown) whereas other zygotes do not appear to stain positive for this modification. (N-P) Staining becomes evident with increasing levels in interphase stage blastomeres at all subsequent developmental stages. (K-M) Weak staining is seen associated with chromatin in cleavage stage metaphase stage blastomere. (Q-S) However, by the blastocyst stage of development the Me(Arg3)H4 modification does appear to partially re-associate with the metaphase stage chromatin (arrows). Fluorescent images represent single confocal sections, therefore only nuclei found within the optical section can be visualized.

View larger version (80K): [in a new window]   Fig. 7. Direct comparison of the levels and localization of specific histone modifications in mouse immature oocyte nuclei and on the metaphase II plate of ovulated eggs. Immature germinal vesicle stage oocytes (GV A-T) and metaphase II arrested eggs (MII A-T) are stained with a panel of histone modification antibodies (Red) and Sytox DNA stain (Green). Transmitted light micrographs are also shown (Light). Regions of DNA that co-localize with histone modifications are seen in yellow (Overlay). Results show that in both immature oocytes and metaphase II arrested eggs, the Me(Lys4)H3 modification strongly co-localizes with DNA (see GV D and MII D). The Me(Lys9)H3 modification also strongly co-localizes with DNA in both immature oocytes and in metaphase II arrested eggs (see GV H and MII H). The hyperacetylated H4 modification also strongly co-localizes with DNA in immature oocyte nuclei (see GV L), however, staining for this modification is negative on metaphase II arrested chromatin (MII K). The Me(Arg17)H3 modification stains strongly in immature oocyte nuclei in a punctate manner (GV O) and appears to co-localize with DNA both around the nucleolus (GV P) and at several other locations in the nucleus (GV P, arrow). Staining for this modification in the ovulated egg, however, is limited to punctate cytoplasmic spots (MII O) and is absent from the chromatin (MII P). Me(Arg3)H4 modification staining is weak and punctate in immature oocyte nuclei (GV S) and does not appear to co-localize with DNA except at one or two locations (GV T, arrow). Staining for this modification in the ovulated egg, however, is absent from the metaphase II chromatin (MII S, MII T). These results show that levels of the hyperacetylated H4, Me(Arg17)H3 and Me(Arg3)H4 modifications are dramatically reduced in ovulated egg chromatin whereas levels of Me(Lys4)H3 and Me(Lys9)H3 modifications do not appear to be affected.

View larger version (39K): [in a new window]   Fig. 8. Treatment of histone H4 with peptidylarginine deiminase results in loss of staining for the arginine 3 histone H4 methyl modification. Histone H4 was purified from 293T cells and treated with either heat-inactivated or active skeletal muscle peptidylarginine deiminase (smPAD) and levels of methylated arginine 3 on histone H4 [ Me(Arg3)H4], hyperacetylated histone H4 ( Hyperacetyl H4), and citrulline (-Mod-Cit) were then detected by western blot analysis. The blots were stained with Ponceau S prior to western analysis and the smPAD and histone H4 (as well as a lower amount of H2A that co-purified with H4) could be visualized. The mobility for H4 and H2A increased following PAD treatment and these modified forms are indicated by asterisks on the Ponceau S stained blot. Staining for the histone Me(Arg3)H4 modification was observed following treatment with heat inactivated smPAD whereas no staining was observed for this modification following treatment with active smPAD. Staining for the hyperacetylated H4 modification appeared to be only slightly reduced (if at all) following smPAD treatment. Citrullination of histone H4 increased dramatically following smPAD treatment. Other contaminating proteins in the H4 sample, including what is probably H2A, were also citrullinated. The citrullinated protein in the heat-inactivated smPAD (control sample) probably indicates that smPAD is capable of deiminating itself. These results provide indirect evidence that the observed loss of staining for methylated arginine residues on egg and embryonic histones was a result of PAD activity.