Báo cáo Y học: Chromatin remodeling in nuclear cloning pptx

Studies involving the modification of chromatin elements such as selective uptake or release of binding proteins, covalent histone modifications including acetylation and methylation, and

M I N I R E V I E W

Chromatin remodeling in nuclear cloning

Paul A Wade1and Nobuaki Kikyo2

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 withdifferentiated adult

nuclei strongly indicate that oocyte cytoplasm contains

unidentified remarkable reprogramming activities withthe

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

I N T R O D U C T I O N

Differentiated somatic nuclei have the flexibility to

dedif-ferentiate in oocyte cytoplasm and redifdedif-ferentiate 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

laevisand 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 suchas 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 eachoocyte 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

E X C H A N G E O F C H R O M A T I N P R O T E I N S Early reports demonstrated that Xenopus or human somatic nuclei injected into Xenopus oocytes lose 80–90% of the preradiolabelled nuclear proteins accompanied withsigni-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 withrecent 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 withDNA 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 suchexample

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 approachagain indicated that nuclear proteins

involved in diverse functions rapidly and constantly

asso-ciate withand dissoasso-ciate 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

H I S T O N E A C E T Y L A T I O N A N D D N A

M E T H Y L A T I O N

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 Suchreprogramming 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 withonly 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 withhyperacetylated 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 caused by aberrant reprogramming of DNA methylation with only potentially subtle abnormalities

H I S T O N E M E T H Y L A T I O N 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

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° withegg 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 withdifferent

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

C O N C L U S I O N S

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 suchas chromo-some domains and DNA function are still open to future investigation Recent technical advances in genomics and systematic analysis of gene expression coupled withan 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

A C K N O W L E D G E M E N T S

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.

HP1 HP1

Me Me Me

Me Me Me

Me HP1

HP1

Me Me

Me

B A

O

H N

CH2

CH2

CH2

CH2 C N

C

C CH 3

H N

CH2

CH2

CH2

CH2 C N

C

C H

H H Methyl lysine

Acetyl lysine

C

Histone Methylation (arginine or lysine)

Nuclear transfer active

demethylation persistent histone exchange

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

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