Báo cáo Y học: Chromatin remodeling in nuclear cloning pptx

MINIREVIEW Chromatin remodeling in nuclear cloning Paul A. Wade 1 and Nobuaki Kikyo 2 1 Department of Pathology, Emory University, Atlanta, GA, USA; 2 Stem Cell Institute and Department of Medicine, University of Minnesota, Minneapolis, MN, USA Nuclear cloning is a procedure to create new animals by injecting somatic nuclei into unfertilized oocytes. Recent successes in mammalian cloning with differentiated adult nuclei strongly indicate that oocyte cytoplasm contains unidentified remarkable reprogramming activities with the capacity to erase the previous memory of cell differentiation. At the heart of this nuclear reprogramming lies chromatin remodeling as chromatin structure and function define cell differentiation through regulation of the transcriptional activities of the cells. Studies involving the modification of chromatin elements such as selective uptake or release of binding proteins, covalent histone modifications including acetylation and methylation, and DNA methylation should provide significant insight into the molecular mechanisms of nuclear dedifferentiation and redifferentiation in oocyte cytoplasm. Keywords: nuclear cloning; chromatin remodeling; linker histone; histone acetylation; histone methylation; DNA methylation. INTRODUCTION Differentiated somatic nuclei have the flexibility to dedif- ferentiate in oocyte cytoplasm and redifferentiate into other multiple lineages during the subsequent embryogenesis. This drastic nuclear plasticity has been repeatedly confirmed by successful nuclear cloning in frogs and several mammalian species over the past half a century (reviewed in [1–3]). Although generally only less than 1% of cloned mice survive to adulthood [1], many of the aborted embryos still contain terminally differentiated tissues derived from the injected somatic nuclei establishing that highly efficient reprogram- ming mechanisms exist in oocyte cytoplasm. Currently, the identities of these activities are ill defined. The toad Xenopus laevis and the mouse represent two of the complementary model organisms for nuclear cloning. Because of its large and abundant oocytes, Xenopus has been used for nuclear cloning since the 1950s, providing a great amount of valuable information regarding a wide range of cell biological and biochemical events that take place in the injected nuclei [2]. On the other hand, mouse cloning is more suitable for genetic studies such as the modification of DNA methylation, genomic imprinting and telomere length. Although these two species display distinct early develop- mental processes, these nuclei share common features upon injection into each oocyte including nuclear swelling, chromatin dispersal and loss of linker histone H1 (see below). In this minireview, we will highlight some of the selected topics on the chromatin modification in Xenopus and mammalian nuclear cloning, followed by a discussion about newly identified histone methylation and heterochro- matin formation as an entirely unexplored field of chroma- tin modification involved in nuclear cloning. EXCHANGE OF CHROMATIN PROTEINS Early reports demonstrated that Xenopus or human somatic nuclei injected into Xenopus oocytes lose 80–90% of the preradiolabelled nuclear proteins accompanied with signi- ficant incorporation of oocyte proteins [2]. Later, exchange of more specific proteins was analyzed in detail. For example, erythrocyte linker histone H1 and H1° are readily replaced with oocyte type linker histone B4, which has lower affinity to linker DNA, by a molecular chaperon, nucleo- plasmin and this contributes to acquisition of transcriptional competence in highly condensed erythrocyte nuclei (Fig. 1A) [4]. Bovine linker histone H1 also becomes undetectable in somatic nuclei after injection into bovine oocytes and it reappears at eight-cell to 16-cell stage, as in normal fertilized embryos [5]. Although nucleoplasmin has not been reported in mammalian oocytes, similar molecules might be involved in the H1 removal in mammalian oocytes as the responsible factor(s) seems to be accumulated in the bovine oocyte nuclei as with Xenopus nucleoplasmin. In contrast, core histones of somatic nuclei are not removed and nucleosomal spacing is not altered when entire chro- matin was tested as bulk [6]. These results are consistent with recent studies using fluorescence recovery after photo- bleaching (FRAP) in living cells. Most of the histone H1 is continuously exchanged between chromatin segments [7] but core histones, especially H3 and H4 are stably associated with DNA in cells [8]. Nonhistone nuclear proteins are also selectively released from or incorporated into somatic chromatin in egg cytoplasm utilizing ATP and GTP [9]. One such example Correspondence to N. Kikyo, Stem Cell Institute and Department of Medicine, University of Minnesota, Mayo Mail Code 716, 420 Delaware St. SE, Minneapolis, MN 55455, USA. Fax: + 1 612 6242436, Tel.: + 1 612 6240498, E-mail: kikyo001@tc.umn.edu Abbreviations: FRAP, fluorescence recovery after photobleaching; TBP, TATA binding protein; ES, embryonic stem cells; PEV, position effect variegation. Dedication: This Minireview Series is dedicated to Dr Alan Wolffe, deceased 26 May 2001. (Received 8 November 2001, accepted 12 February 2002) Eur. J. Biochem. 269, 2284–2287 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02887.x is the basal transcription factor TATA binding protein (TBP) that is released from somatic chromatin by a chromatin remodeling protein complex containing ISWI, a member of the SWI2/SNF2 superfamily (Fig. 1B). A FRAP approach again indicated that nuclear proteins involved in diverse functions rapidly and constantly asso- ciate with and dissociate from nuclear infrastructure in the physiological state [10]. Overall, these results suggest the exchange of chromatin proteins between somatic nuclei and oocyte cytoplasm are reminiscence of the physiological protein exchange that occurs in intact living cells. Because of the extreme difference of differentiation status between somatic cells and oocytes, these protein exchange events give rise to more drastic and easily detectable consequences leading to dedifferentiation. HISTONE ACETYLATION AND DNA METHYLATION Importantly, these chromatin remodeling events can be accomplished in the absence of DNA replication accom- panied with nucleosomal disassembly, which implies that more specific mechanisms are actively engaged in repro- gramming somatic nuclei. Somatic nuclei transplanted into Xenopus oocytes do not replicate genomic DNA, unlike those transplanted into eggs (oocytes after ovulation are called eggs in Xenopus.), but they transcribe genes very actively. Oocyte cytoplasm can modify the transcriptional pattern of injected somatic nuclei into an oocyte pattern. Such reprogramming of transcriptional activity has been clearly described in the two types of 5S rRNA genes in Xenopus. While the oocyte 5S rRNA gene is transcribed only in oocytes and early embryos through gastrula, the somatic type is detectable in most cells. When neurula nuclei with only the somatic 5S genes active were injected into oocytes, the inactive oocyte type gene was reactivated followed by a normal pattern of inactivation at gastrulation, which recapitulated the normal developmental expression profile [11,12]. In somatic cells, transcriptionally active somatic 5S rRNA genes are packaged with hyperacetylated histone H4 but the silent oocyte types are associated with hypoacetylated histone H4 [13] suggesting that histone H4 of the oocyte type 5S genes of somatic nuclei might become acetylated in oocyte cytoplasm, facilitating their transcrip- tional activation. Histone acetylation and DNA methylation are tightly coupled through protein complexes containing DNA meth- yltransferase 1 and histone deacetylases [14,15]. Reprogram- ming of DNA methylation following nuclear transfer appears inefficient as cloned bovine blastocysts demonstra- ted DNA methylation patterns more similar to donor cells in various genomic regions than to normal blastocysts [16]. In addition, individual blastocysts displayed significant varia- tions in the degree of methylation. Surprisingly, such aberrant reprogramming of DNA methylation does not necessarily have devastating effects on mouse cloning. Mouse clones derived from embryonic stem (ES) cell nuclei, which showed extremely unstable and variable methylation pat- terns prior to injection into oocytes, demonstrated higher survival rate to adulthood than any other types of somatic nuclei [17]. This result implies that mouse clones can tolerate the genetic noise causedbyaberrant reprogramming of DNA methylation with only potentially subtle abnormalities. HISTONE METHYLATION Eukaryotic genomes are functionally compartmentalized into active and inactive fractions. A classic example of genome partitioning results from the action of genes referred to as suppressors of variegation, or Su(var)s. Position effect variegation, or PEV, results when an euchromatic gene, through chromosomal translocation, is placed in proximity to a heterochromatic region (reviewed in [18]). Cells express the translocated euchromatic gene in a mosaic pattern despite the lack of any mutations in the gene itself. Genetic screens have identified a set of factors with the ability to modify variegation when mutated. These genes fall into two classes: suppressors of PEV [Su(var)s] are genes where mutation leads to an increased expression of variegated genes, and enhancers have the opposite effect when mutated. The suppressor class includes such factors as histone deacetylases and structural components of hetero- chromatin such as HP1 (reviewed in [19]). Elegant work from T. Jenuwein and colleagues has recently assigned an enzymatic activity to a member of this class, a human homolog of the Su(var)3–9 protein of Drosophila, known as SUV39H1. SUV39H1, and its homologues in other organisms, catalyze the addition of a methyl group to the epsilon amino group of lysine 9 in N N N N H1 A B Nucleoplasmin Stable chromatin Less stable chromatin N N TBP N N ISWI complex B4 H1 B4 TBP More relaxed chromatin Fig. 1. Two diagrammatic examples of chromatin remodeling in Xenopus egg cytoplasm. (A) Nucleoplasmin exchanges somatic linker histones H1 and H1° with egg linker B4 leading to less stable chro- matin that is more favorable for active transcription and DNA repli- cation in early embryos. N, nucleosome. (B) Chromatin remodeling ISWI complex relaxes DNA–histone interactions, which induces the release of TBP from DNA. Ó FEBS 2002 Chromatin remodeling in nuclear cloning (Eur. J. Biochem. 269) 2285 histone H3 [20]. This lysine residue is absolutely conserved through evolution. Its location at the N-terminus of histone H3, outside the histone-fold motif, potentially places this residue in an exposed, accessible position on the nucleosome [21]. Covalent modification of this lysine could, in theory, result in either a change in the biophysical properties of chromatin or it could provide regulatory information. Unlike lysine acetylation, methylation does not result in neutralization of the positive charge associated with the amino group. While changes in acetylation state of the core histone N-termini have been conclusively linked to changes in the compaction properties of model chromatin fibers [22], no such data exists for lysine methylation. The alternative model, that specific patterns of histone modification form favorable binding sites for nonhistone chromatin proteins, has been termed the Ôhistone codeÕ hypothesis [23]. In the case of lysine methylation, the independent finding by two different groups that histone H3 methylated at lysine 9 forms a preferential binding site for the heterochromatin compo- nent HP1 [24,25] provides strong support for the histone code hypothesis. The association of HP1 proteins with histone H3 methylated at lysine 9 suggests that this covalent histone modification provides a stable epigenetic mark for transcriptionally repressed chromatin domains (Fig. 2A). How might such covalent modification of core histones affect the outcome of nuclear transfer? Some covalent modifications of the core histones, such as acetylation and phosphorylation, are freely reversible. Enzymes that catalyze these reactions exist and are well studied. Methylation of lysine and arginine residues constitutes a somewhat different challenge. Histones are known to be heavily modified by methylation of arginine and lysine residues, with the modification of specific sites associated with different functional roles [26]. In contrast to acetylation, lysine methylation of histones is known to be a quite stable modification [27]. The chemistry of a tetrahedral methyl carbon in methyl lysine differs substantially from the planar carbonyl carbon of acetyl lysine (Fig. 2B). In fact, it is currently unknown whether lysine or arginine methylation is reversible. One can imagine several possible fates for histones methylated at lysine and arginine residues in nuclear transfer experiments (Fig. 2C). If demethylase enzymes exist, then these enzymes may erase the epigenetic mark of histone methylation following transplantation of the somatic nuclei into oocytes. Alternatively, it is conceivable that a subset of the core histones (including perhaps those marked by methylation) may be exchanged from the chromosomes following injection into the recipient oocyte, or that the modified lysine residue may be removed from the histone by proteolysis. Finally, it is entirely possible that histone methylation represents a permanent epigenetic mark that persists following transfer of somatic nuclei into oocytes. The functional consequences of persistence of such an epigenetic mark have so far not been explored. Deciphering the developmental appearance of histone methylation, the reversibility of this modification, and whether erasure of this epigenetic mark impacts the outcome of nuclear transfer remain important challenges for the future. CONCLUSIONS Our understanding of the mechanisms and roles of chro- matin remodeling in nuclear cloning is still in its infancy. The relationships of chromatin structure, ranging from the most fundamental level (nucleosomal positioning and nuclease sensitivity) to more global issues such as chromo- some domains and DNA function are still open to future investigation. Recent technical advances in genomics and systematic analysis of gene expression coupled with an increased emphasis on chromatin architecture promise new possibilities in the comparison of transcriptional profiles of cloned vs. natural embryos. Improved understanding of the molecular mechanisms of nuclear reprogramming will potentially lead to an enhanced ability to engineer cells with desired traits for therapeutic purposes without the use of human embryonic materials. ACKNOWLEDGEMENTS P. W. gratefully acknowledges financial support from the National Institute of Child Health and Human Development, from the Rett Syndrome Research Foundation, and from the Massachusetts Rett Syndrome Association. HP1HP1 Me Me Me Me Me Me Me HP1 Me HP1 HP1 Me Me Me B A O H N CH 2 CH 2 CH 2 CH 2 CH N C C CH 3 H N CH 2 CH 2 CH 2 CH 2 CH N C C H H H Methyl lysine Acetyl lysine C Histone Methylation (arginine or lysine) Nuclear transfer active demethylation histone exchange persistent methylation H + proteolysis of histone N-terminus Fig. 2. Structure and function of histone H3 methylation. (A) The cartoon depicts a transcriptionally repressed chromatin domain. Methylation of histone H3 at lysine 9 (red circles) leads to recruit- ment of HP1 (blue squares). The resulting chromatin condensation contributes to the repressed state. (B) Chemical structure of mono- methyl lysine and acetyl lysine. The carbon atom bonded to the nitrogen atom of the epsilon amino group differs substantially in the two forms of lysine modification. In monomethyl lysine, the nitrogen is bonded to a tetrahedral methyl carbon and positive charge is maintained. In acetyl lysine, the nitrogen forms an amide bond with the carbonyl carbon of the acetyl group resulting in neutralization of the positive charge. (C) The flow diagram depicts the potential fates of methylated histones following nuclear transfer. Erasure of the epigenetic mark can occur either through active demethylation, through proteolysis of the histone N-terminus, or through core his- tone exchange. Alternatively, the methyl lysine may persist resulting in propagation of an epigenetic state. 2286 P. A. Wade and N. Kikyo (Eur. J. Biochem. 269) Ó FEBS 2002 REFERENCES 1. Solter, D. (2000) Mammalian cloning: advances and limitations. Nat. Rev. Genet. 1, 199–207. 2. Gurdon, J.B., Laskey, R.A., De Robertis, E.M. & Partington, G.A. (1979) Reprogramming of transplanted nuclei in amphibia. Int. Rev. Cytol. S9, 161–178. 3. Rideout, I.W., Eggan, K. & Jaenisch, R. (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293, 1093– 1098. 4. Dimitrov, S. & Wolffe, A.P. 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