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Linker Histone H1 in Early Mouse Embryogenesis 2899 Journal of Cell Science 113, 2897-2907 (2000) 2897 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1314 Somatic linker histone H1 is present throughout mouse embryogenesis and is not replaced by variant H1° Pierre G. Adenot1,*, Evelyne Campion1, Edith Legouy1, C. David Allis2, Stefan Dimitrov3, Jean-Paul Renard1 and Eric M. Thompson1,4 1Unité de Biologie du Développement, Institut National de la Recherche Agronomique, F-78352 Jouy-en-Josas, France 2Department of Biochemistry and Molecular Genetics, University of Virginia Health Science Center, Charlottesville, Virginia 22908, USA 3Laboratoire de Biologie Moléculaire et Cellulaire de la Différenciation, INSERM U 309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche, Cedex, France 4Sars International Center, Bergen High Technology Center, Thormøhlensgt. 55, N-5008 Bergen, Norway *Author for correspondence (e-mail: [email protected]) Accepted 9 June; published on WWW 20 July 2000 SUMMARY A striking feature of early embryogenesis in a number of envelopes, somatic H1 was rapidly incorporated onto organisms is the use of embryonic linker histones or high maternal and paternal chromatin, and the amount of mobility group proteins in place of somatic histone H1. The somatic H1 steadily increased on embryonic chromatin transition in chromatin composition towards somatic H1 through to the 8-cell stage. Microinjection of somatic appears to be correlated with a major increase in H1 into oocytes, and nuclear transfer experiments, transcription at the activation of the zygotic genome. demonstrated that factors in the oocyte cytoplasm and the Previous studies have supported the idea that the mouse nuclear envelope, played central roles in regulating the embryo essentially follows this pattern, with the significant loading of H1 onto chromatin. Exchange of H1 from difference that the substitute linker histone might be the transferred nuclei onto maternal chromatin required differentiation variant H1°, rather than an embryonic breakdown of the nuclear envelope and the extent of variant. We show that histone H1° is not a major linker exchange was inversely correlated with the developmental histone during early mouse development. Instead, somatic advancement of the donor nucleus. H1 was present throughout this period. Though present in mature oocytes, somatic H1 was not found on maternal Key words: Histone H1°, Genome activation, Oocyte, Nuclear metaphase II chromatin. Upon formation of pronuclear transfer INTRODUCTION up and down regulation of the expression of specific genes (Shen et al., 1995; Shen and Gorovsky, 1996; Dou et al., 1999). Linker histones interact with spacer DNA between adjacent A potentially regulatory facet in a number of organisms is the nucleosomal histone octamer cores. The traditional view that use of a repertoire of linker histone variants which differ both they are a stoichiometric structural component of chromatin, in their globular domains, and in modification of the length with an essentially repressive role in regulating transcription, and net charge of the C-terminal domain. In the mouse, for has been undergoing revision. In contrast to the structural example, there are five somatic variants H1a, H1b, H1c, H1d and sequence conservation of the core histones, there is and H1e, a testis specific variant H1t, and the variant H1° considerable divergence in both sequence and structure among which is expressed only in some lineages of differentiated cells linker histones. Metazoan linker histones contain a central (Franke et al., 1998). In the transition from oocyte to somatic globular domain with N- and C-terminal tails, but the 5S rRNA gene expression during Xenopus embryogenesis protozoan, Tetrahymena, has a linker histone which contains (Wolffe, 1989; Bouvet et al., 1994), somatic histone H1 binds only the C-terminal tail (reviewed by Wolffe et al., 1997). The equally to both oocyte and somatic 5S nucleosomal templates C-terminal region is rich in basic amino acids, and it is likely (Howe et al., 1998) but it selectively represses the oocyte that this tail domain interacts with negatively charged linker template through binding to the 3′ end of the nucleosomal DNA to facilitate chromatin condensation (Ramakrishnan, core, resulting in stable positioning of a nucleosome over key 1997). regulatory elements (Sera and Wolffe, 1998). On the somatic The knockout of histone H1 in Tetrahymena revealed two template, H1 binds to the 5′ end of the nucleosomal core, important points; histone H1 is not essential for nuclear leaving key promoter elements accessible. In the chicken, assembly or cell survival, and it appears to be involved in both where the six H1 genes encode different H1 protein sequences 2898 P. G. Adenot and others (Nakayama et al., 1993), different protein patterns were show that somatic histone H1 was present in chromatin until obtained from a series of mutants cell lines, each lacking one nuclear breakdown during meïotic oocyte maturation, and then of the H1 genes, indicating that H1 variants may play distinct reassembled onto chromatin following pronuclear formation at roles in the transcriptional regulation of specific genes (Takami the onset of embryogenesis. Histone H1° was not detected on et al., 2000). chromatin during this developmental period. The absence of A particular feature of early embryogenesis in some animals, H1 on maternal metaphase II chromosomes, contrasted with its is the absence of somatic linker histones during the initial presence on chromosomes at the first mitosis and a weak cleavage stages. Prior to the mid-blastula transition (MBT) in presence in sperm chromatin. Microinjection of somatic H1 Xenopus embryos, an embryonic variant H1M (or B4) replaces into oocytes did not result in staining of maternal somatic H1 (Smith et al., 1988; Dimitrov et al., 1993). The high chromosomes, but in nuclear transfer experiments, we mobility group protein HMG-1, together with the B4 linker observed that somatic H1 could be loaded onto maternal histone, are major components of chromatin within the nuclei chromosomes only when the transferred nucleus lost its nuclear assembled during the incubation of Xenopus sperm chromatin membrane and formed prematurely condensed chromosomes in Xenopus egg extract (Nightingale et al., 1996). Both proteins (PCC). The extent of transfer of H1 to maternal chromosomes, bind to linker DNA but less tightly than somatic H1 (Ura et al and its removal from PCC, appeared to be mediated by 1996), and thus may facilitate rapid cycles of DNA replication. factors in the oocyte cytoplasm, but also depended on the In Drosophila embryos, an HMG-1 homologue, HMG-D, developmental stage of the transferred nucleus. replaces somatic H1 until the MBT (Ner and Travers, 1994). In the sea urchin, maternal cleavage stage histones are present until the third cell cycle, and the cleavage stage linker histone MATERIALS AND METHODS has a high homology to Xenopus histone B4 (Mandl et al., 1997). However, it remains questionable whether this feature Collection of embryos and oocytes can be extended to early embryogenesis in general, or if it may Female C57/CBA mice, 6-8 weeks old, were superovulated with instead reflect a situation in which rapid DNA replication intraperitoneal injections of 5 i.u. of pregnant mare serum (PMS; occurs in the absence of transcription from the zygotic genome. Folligon, Intervet), followed 46-48 hours later with 5 i.u. human In the mouse, the initial observation by Clarke et al. (1992), chorionic gonadotropin (hCG; Chlorulon, Intervet). To recover mature oocytes in metaphase II (MII), superovulated females were sacrificed that somatic histone H1 is first detected cytochemically in a 15 hours post-hCG (phCG). To obtain fertilized oocytes, females were portion of embryos at the 4-cell stage and by the 8-cell stage caged with C57/CBA males immediately after hCG injection, and in all nuclei in all embryos, suggested that the mouse embryo embryos were collected at the one-cell stage. Oocytes and embryos followed the early cleavage pattern of the sea urchin, Xenopus were incubated after collection in 0.5% hyaluronidase (Sigma) in PB1 and Drosophila, in maintaining the absence of a somatic linker for 1-2 minutes at 37°C to remove cumulus cells, washed extensively histone. The mouse embryo, however, would deviate in two in PB1, and then returned to culture in M16 medium under 5% CO2 important ways; somatic H1 appears one full cell cycle after in air, until fixation. To recover immature oocytes at prophase I, major activation of the zygotic genome (reviewed by Latham, female C57/CBA mice, 11-13 weeks old, were superovulated with 1999), and Clarke et al. (1997) subsequently proposed that the intraperitoneal injections of PMS as described above. Ovaries were differentiation variant H1°, rather than an embryonic variant, removed 45 to 50 hours after injection, and transferred to PB1 prewarmed at 37°C. Fully grown oocytes were freed from peripheral was the substitute linker histone. In the ovary, the fully grown cells by gentle pipetting, and either fixed immediately, or returned to oocyte is a highly specialized cell which results from a culture to be fixed during nuclear maturation. differentiation process during oogenesis. From this point of view, the presence of the differentiation variant H1° in Nuclear transfer procedures chromatin could be considered consistent. The amount of H1° Hybrid cells were reconstructed by electrofusion between MII oocytes increases during terminal cell differentiation
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