Introduction Upon ovulation, a terminally differentiated mammalian oocyte will die if it does not bind and fuse with a sperm (Dean et al., 2003). If fertilization does occur, maternal gene products orchestrate the transformation of the egg into a totipotent zygote within hours. This reprogramming process is associated with dramatic alterations of protein synthesis patterns between the zygotic and two-cell embryo stage (Cullen et al., 1980; Levinson et al., 1978). Dynamic changes in the local and global organization of chromatin are surfacing as key regulators of genomic function and there is now considerable evidence that many of these changes are regulated epigenetically (Hansen et al., 1998; Nakao, 2001). When considering the role of epigenetics in embryonic reprogramming events, DNA methylation at the symmetrical dinucleotide CpG by DNA methyltransferase has received much attention (Pickard et al., 2001). Post-translational modification of core histone proteins, however, represents another attractive epigenetic mark that will probably also require reprogramming in germ cells and the early embryo. It is well established that specific amino acid residues within histone proteins are targets for a number of post-translational modifications including acetylation, phosphorylation, methylation, poly (ADP-ribosylation), and ubiquitination (Fischle et al., 2003). These covalent histone modifications provide an attractive storage mechanism for mitotically- and meiotically inheritable information that can be ‘read’ by various effector proteins. Thus, by regulating access to underlying DNA, histone modifications and effector proteins may dictate correct spatial and temporal gene expression patterns during development (Jenuwein and Allis, 2001). These modifications can be read by transcriptional machinery in a sequential or combinatorial fashion and thus can give rise to various patterns of expression that are dynamic, complex, and specific (Strahl and Allis, 2000). Although not rigorously tested, it has long been predicted that, during embryonic reprogramming, specific histone modifications are removed from the chromatin template and new marks are placed onto different regions of chromatin that facilitate expression of the embryonic genome (Li, 2002). Here, we investigate this problem at a global level using a wide range of modification-selective antibodies. By documenting a collection of remarkable and highly reproducible changes during oocyte maturation and early embryonic development, our findings lend support to the histone modification ‘resetting’ prediction. Of particular relevance to animal cloning endeavors, our results suggest that the egg cytoplasm may contain enzymatic activities that are capable of removing both acetyl and arginine methyl modifications from specific residues within histone proteins. 4449 In order to investigate whether covalent histone modifications may be involved in early embryonic reprogramming events, changes in global levels of a series of histone tail modifications were studied during oocyte maturation and pre-implantation mouse development using indirect immunofluorescence and scanning confocal microscopy. Results showed that histone modifications could be classified into two strikingly distinct categories. The first contains stable ‘epigenetic’ marks such as histone H3 lysine 9 methylation [Me(Lys9)H3], histone H3 lysine 4 methylation [Me(Lys4)H3] and histone H4/H2A serine 1 phosphorylation [Ph(Ser1)H4/H2A]. The second group contains dynamic and reversible marks and includes hyperacetylated histone H4, histone H3 arginine 17 methylation [Me(Arg17)H3] and histone H4 arginine 3 methylation [Me(Arg3)H4]). Our results also showed that removal of these marks in eggs and early embryos occurs during metaphase suggesting that the enzymes responsible for the loss of these modifications are probably cytoplasmic in nature. Finally, we provide data demonstrating that treatment of cellular histones with peptidylarginine deiminase (PAD) results in loss of staining for the histone H4 arginine 3 methyl mark, suggesting that PADs can reverse histone arginine methyl modifications. Supplemental data available online at http://jcs.biologists.org/cgi/ content/full/117/19/4449/DC1 Key words: Epigenetics, Histone modifications, Oocyte, Early embryo Summary Dynamic alterations of specific histone modifications during early murine development Olga F. Sarmento 1 , Laura C. Digilio 1 , Yanming Wang 2 , Julie Perlin 3 , John C. Herr 1 , C. David Allis 2 and Scott A. Coonrod 1,3, * 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 *Author for correspondence (e-mail: scc2003@med.cornell.edu) Accepted 25 May 2004 Journal of Cell Science 117, 4449-4459 Published by The Company of Biologists 2004 doi:10.1242/jcs.01328 Research Article