DNA Methylation patterns and epigenetic memory

10.1101/gad.947102Access the most recent version at doi: 2002 16: 6-21

Genes Dev.

Adrian Bird

DNA methylation patterns and epigenetic memory

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REVIEW

DNA methylation patterns and epigenetic memory

Adrian Bird1

Wellcome Trust Centre for Cell Biology,University of Edinburgh,Edinburgh EH93JR,UK

The character of a cell is defined by its constituent pro-teins,which are the result of specific patterns of gene expression.Crucial determinants of gene expression pat-terns are DNA-binding transcription factors that choose genes for transcriptional activation or repression by rec-ognizing the sequence of DNA bases in their promoter regions.Interaction of these factors with their cognate sequences triggers a chain of events,often involving changes in the structure of chromatin,that leads to the assembly of an active transcription complex(e.g.,Cosma et al.1999).But the types of transcription factors present in a cell are not alone sufficient to define its spectrum of gene activity,as the transcriptional potential of a ge-nome can become restricted in a stable manner during development.The constraints imposed by developmen-tal history probably account for the very low efficiency of cloning animals from the nuclei of differentiated cells (Rideout et al.2001;Wakayama and Yanagimachi2001). A“transcription factors only”model would predict that the gene expression pattern of a differentiated nucleus would be completely reversible upon exposure to a new spectrum of factors.Although many aspects of expres-sion can be reprogrammed in this way(Gurdon1999), some marks of differentiation are evidently so stable that immersion in an alien cytoplasm cannot erase the memory.

The genomic sequence of a differentiated cell is thought to be identical in most cases to that of the zy-gote from which it is descended(mammalian B and T cells being an obvious exception).This means that the marks of developmental history are unlikely to be caused by widespread somatic mutation.Processes less irrevocable than mutation fall under the umbrella term “epigenetic”mechanisms.A current definition of epige-netics is:“The study of mitotically and/or meiotically heritable changes in gene function that cannot be ex-plained by changes in DNA sequence”(Russo et al. 1996).There are two epigenetic systems that affect ani-mal development and fulfill the criterion of heritability: DNA methylation and the Polycomb-trithorax group (Pc-G/trx)protein complexes.(Histone modification has some attributes of an epigenetic process,but the issue of heritability has yet to be resolved.)This review concerns DNA methylation,focusing on the generation,inheri-tance,and biological significance of genomic methyl-ation patterns in the development of mammals.Data will be discussed favoring the notion that DNA methyl-ation may only affect genes that are already silenced by other mechanisms in the embryo.Embryonic transcrip-tion,on the other hand,may cause the exclusion of the DNA methylation machinery.The heritability of meth-ylation states and the secondary nature of the decision to invite or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory function in development.Indeed,the possibility will be discussed that DNA methylation and Pc-G/trx may rep-resent alternative systems of epigenetic memory that have been interchanged over evolutionary time.Animal DNA methylation has been the subject of several recent reviews(Bird and Wolffe1999;Bestor2000;Hsieh2000; Costello and Plass2001;Jones and Takai2001).For re-cent reviews of plant and fungal DNA methylation,see Finnegan et al.(2000),Martienssen and Colot(2001),and Matzke et al.(2001).

Variable patterns of DNA methylation in animals

A prerequisite for understanding the function of DNA methylation is knowledge of its distribution in the ge-nome.In animals,the spectrum of methylation levels and patterns is very broad.At the low extreme is the nematode worm Caenorhabditis elegans,whose genome lacks detectable m5C and does not encode a conven-tional DNA methyltransferase.Another invertebrate, the insect Drosophila melanogaster,long thought to be devoid of methylation,has a DNA methyltransferase-like gene(Hung et al.1999;Tweedie et al.1999)and is reported to contain very low m5C levels(Gowher et al. 2000;Lyko et al.2000),although mostly in the CpT di-nucleotide rather than in CpG,which is the major target for methylation in animals.Most other invertebrate ge-nomes have moderately high levels of methyl-CpG con-centrated in large domains of methylated DNA separated by equivalent domains of unmethylated DNA(Bird et al. 1979;Tweedie et al.1997).This mosaic methylation pat-tern has been confirmed at higher resolution in the sea

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squirt,Ciona intestinalis(Simmen et al.1999).At the opposite extreme from C.elegans are the vertebrate ge-nomes,which have the highest levels of m5C found in the animal kingdom.Vertebrate methylation is dis-persed over much of the genome,a pattern referred to as global methylation.The variety of animal DNA methyl-ation patterns highlights the possibility that different distributions reflect different functions for the DNA methylation system(Colot and Rossignol1999). Mammalian DNA methylation patterns vary in time and space

In human somatic cells,m5C accounts for～1%of total DNA bases and therefore affects70%–80%of all CpG dinucleotides in the genome(Ehrlich1982).This average pattern conceals intriguing temporal and spatial varia-tion.During a discrete phase of early mouse develop-ment,methylation levels in the mouse decline sharply to～30%of the typical somatic level(Monk et al.1987; Kafri et al.1992).De novo methylation restores normal levels by the time of implantation.A much more limited drop in methylation occurs in the frog Xenopus laevis (Stancheva and Meehan2000),and no drop is seen in the zebrafish,Danio rerio(MacLeod et al.1999).Even within vertebrates,therefore,interspecies variation is seen that could reflect differences in the precise role played by methylation in these organisms.For mice and probably other mammals,however,the cycle of early embryonic demethylation followed by de novo methylation is criti-cal in determining somatic DNA methylation patterns.

A genome-wide reduction in methylation is also seen in primordial germ cells(Tada et al.1997;Reik et al.2001) during the proliferative oogonial and spermatogonial stages.

The most striking feature of vertebrate DNA methyl-ation patterns is the presence of CpG islands,that is, unmethylated GC-rich regions that possess high relative densities of CpG and are positioned at the5?ends of many human genes(for review,see Bird1987).Compu-tational analysis of the human genome sequence pre-dicts29,000CpG islands(Lander et al.2001;Venter et al. 2001).Earlier studies estimated that～60%of human genes are associated with CpG islands,of which the great majority are unmethylated at all stages of develop-ment and in all tissue types(Antequera and Bird1993). Because many CpG islands are located at genes that have a tissue-restricted expression pattern,it follows that CpG islands can remain methylation-free even when their associated gene is silent.For example,the tissue-specifically expressed human?-globin(Bird et al.1987) and?2(1)collagen(McKeon et al.1982)genes have CpG islands that remain unmethylated in all tested tissues, regardless of expression.

A small but significant proportion of all CpG islands become methylated during development,and when this happens the associated promoter is stably silent.Devel-opmentally programmed CpG-island methylation of this kind is involved in genomic imprinting and X chromo-some inactivation(see below).The de novo methylation events occur in germ cells or the early embryo(Jaenisch et al.1982),suggesting that de novo methylation is par-ticularly active at these stages.There is evidence,how-ever,that de novo methylation can also occur in adult somatic cells.A significant fraction of all human CpG islands are prone to progressive methylation in certain tissues during aging(for review,see Issa2000),or in ab-normal cells such as cancers(for review,see Baylin and Herman2000)and permanent cell lines(Harris1982;An-tequera et al.1990;Jones et al.1990).The rate of accu-mulation of methylated CpGs in somatic cells appears to be very slow.For example,de novo methylation of a provirus in murine erythroleukemia cells took many weeks to complete(Lorincz et al.2000).Similarly,the recovery of global DNA methylation levels following chronic treatment of mouse cells with the DNA meth-ylation inhibitor5-azacytidine required months(Flatau et al.1984).

How do patterns of methylated and unmethylated mammalian DNA arise in development and how are they maintained?Why are CpG islands usually,but not always,methylation-free?What causes methylation of bulk non-CpG-island DNA?These burning questions cannot be answered definitively at present,but there are distinct hypotheses that have been addressed experimen-tally.The available data will be conveniently considered in three parts:(1)mechanisms for maintaining DNA methylation patterns;(2)mechanisms and consequences of methylation gain;and(3)mechanisms and conse-quences of methylation loss.

Maintenance methylation—not so simple Maintenance methylation describes the processes that reproduce DNA methylation patterns between cell gen-erations.The simplest conceivable mechanism for main-tenance depends on semiconservative copying of the pa-rental-strand methylation pattern onto the progeny DNA strand(Holliday and Pugh1975;Riggs1975).In keeping with the model,the methylating enzyme DNMT1prefers to methylate those new CpGs whose partners on the parental strand already carry a methyl group(Bestor1992;Pradhan et al.1999).Thus a pattern of methylated and nonmethylated CpGs along a DNA strand tends to be copied,and this provides a way of passing epigenetic information between cell generations. The idea that mammalian DNA methylation patterns are established in early development by de novo meth-yltransferases DNMT3A and DNMT3B(Okano et al. 1998a,1999;Hsieh1999b)and then copied to somatic cells by the maintenance DNA methyltransferase DNMT1is elegant and simple,but,as discussed below, may not fully explain persistence of methylation pat-terns during cell proliferation.

Experiments that first showed replication of methyl-ation patterns on artificially methylated DNA also re-vealed a relatively low fidelity for the process(Pollack et al.1980;Wigler et al.1981).After many cell generations, methylation of the introduced DNA was retained,but at a much lower level than in the starting plasmid.The

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failure of maintenance was estimated to occur with a frequency of～5%per CpG site per cell division.Quan-titative studies of an endogenous CpG site broadly agreed with this figure(Riggs et al.1998).Cell clones in which this site was initially unmethylated acquired methylation and clones where it was methylated lost methylation.The rate of change was estimated at～4% per cell generation.Error rates of this magnitude mean that a detailed methylation pattern would eventually be-come indistinct as cells proliferate.Indeed,dynamic changes in detailed methylation patterns have been ob-served in monoclonal lyomyomas(Silva et al.1993)and at the methylated FMR1gene(St?ger et al.1997).These studies established that clonal populations of cells do not have the homogeneous methylation patterns that would be predicted by the replication model of mainte-nance methylation.Not only does DNA methyltransfer-ase fail to complete half-methylated sites at a significant rate,but also significant de novo methylation occurs at unmethylated sites.

At first sight,these findings appear to undermine the concept of maintenance methylation,but this does not follow.Although detailed methylation patterns may not be maintained at the level of a single CpG nucleotide, the methylation status of DNA domains appears to be faithfully propagated during development(Pfeifer et al. 1990).CpG islands,for example,keep their overall un-methylated state(or methylated state)extremely stably through multiple cell generations.DNMT1is partly re-sponsible for this stability,but there is likely to be an-other as yet unknown component to the maintenance process.Dramatic evidence for this alternative mainte-nance mechanism comes from the finding that CpG-is-land methylation is stably maintained even in the appar-ent absence of the only known maintenance DNA meth-yltransferase,DNMT1(Rhee et al.2000).A similar phenomenon may account for the maintenance of allele-specific DNA methylation imprints under conditions where the concentration of DNMT1is severely limiting (Jaenisch1997).

De novo DNA methylation by default?

The origin of DNA methylation patterns is a long-stand-ing mystery in the field.The de novo methyltransferases DNMT3A and DNMT3B(Okano et al.1998a,1999)are highly expressed in early embryonic cells,and it is at this stage that most programmed de novo methylation events occur.What determines which regions of the genome should be methylated?An extreme possibility is that de novo DNA methylation in early mammalian develop-ment is an indiscriminate process potentially affecting all https://www.wendangku.net/doc/c01120385.html,patible with the default model is the ap-parent absence of intrinsically unmethylatable DNA se-quences in mammalian genomes.Even CpG islands, most of which are unmethylated at all times in normal cells,can acquire methylation under special develop-mental circumstances or in abnormal cells(permanent cell lines or cancer cells).It is clear,however,that not all regions of the genome are equally accessible to DNA methyltransferases.DNMT3B in particular is known to be required for de novo methylation of specific genomic regions,as mice or human patients with DNMT3B mu-tations are deficient in methylation of pericentromeric repetitive DNA sequences and at CpG islands on the inactive X chromosome(Miniou et al.1994;Okano et al. 1998b;Hansen et al.2000;Kondo et al.2000).DNMT3B may therefore be adapted to methylate regions of silent chromatin.

Evidence that accessory factors are also needed to en-sure appropriate methylation came initially from plants, where the SNF2-like protein DDM1was shown to be essential for full methylation of the Arabidopsis thaliana genome(Jeddeloh et al.1999).An equivalent dependence is seen in animals,as mutations in human ATRX(Gibbons et al.2000)and mouse Lsh2genes(Den-nis et al.2001),both of which encode relatives of the chromatin-remodeling protein SNF2,have significant ef-fects on global DNA methylation patterns.Loss of LSH2 protein,in particular,matches the phenotype of the DDM1mutation in Arabidopsis,for both mutants lose methylation of highly repetitive DNA sequences,but re-tain some methylation elsewhere in the genome.Per-haps efficient global methylation of the genome requires perturbation of chromatin structure by these chromatin-remodeling proteins so that DNMTs can gain access to the DNA.Collaboration between DNMTs and factors that allow them access to specialized chromosomal re-gions may be particularly important in regions that are heterochromatic and inaccessible.Although the net re-sult of these processes is apparently global genomic methylation,the evidence for selectivity means that the word“default”is probably not appropriate.

Targeting de novo methylation to preferred

DNA sequences

Another hypothesis to explain global methylation is that the DNA methylation machinery is preferentially at-tracted by certain DNA sequences in the mammalian genome(Turker1999).The presence of high levels of methylation in DNA outside such a DNA methylation center could be explained by spreading into the sur-rounding DNA.Barriers to spreading would lead to the formation of CpG islands.A hypothetical trigger for DNA methylation is DNA sequence repetition,which can promote de novo methylation in filamentous fungi and plants under certain circumstances(Selker1999; Martienssen and Colot2001).The most suggestive evi-dence in mammals concerns manipulation of transgene copy number at a single locus in the mouse genome us-ing cre-lox technology(Garrick et al.1998).High levels of transgene repetition were found to cause significant transgene silencing and concomitant methylation.The efficiency of expression increased as copy number was reduced at the locus,and the level of methylation de-creased.Whether repetition caused methylation directly, or indirectly as a consequence of some other event(e.g., transcriptional silencing;see below),is not known. The clearest definition of a DNA methylation center

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comes from the fungus Neurospora,where short TpA-rich segments of DNA were found to induce methylation (Miao et al.2000).Identification of a mammalian DNA methylation center located upstream of the mouse ad-enine phophoribosyltransferase(APRT)gene has been re-ported(Mummaneni et al.1993;Yates et al.1999).The region contains B1repetitive elements and attracts high levels of de novo methylation upon transfection into em-bryonic cells,although the effect is relative,because many DNA sequences are subject to de novo methyl-ation in these cells.The APRT methylation center be-comes methylated in DNMT1-deficient ES cells,sup-porting the idea that it corresponds to a region that is a favorable substrate for de novo methylation(Yates et al. 1999).

Because the evidence suggests that replication of methylation patterns by DNMT1is only partly respon-sible for maintenance methylation(see above),an attrac-tive possibility is that the features of a DNA domain that help maintain its methylated status are the same fea-tures that promote its de novo methylation.Imprinting boxes,for example,whose differential methylation is as-sociated with genomic imprinting(Tremblay et al.1997; Birger et al.1999;Shemer et al.2000),tend to retain their methylation levels tenaciously even when the amount of the maintenance enzyme DNMT1is reduced(Beard et al.1995).The de novo methylases DNMT3A and DNMT3B(Okano et al.1998a,1999)may be attracted disproportionately to these sequences,and this attrac-tion may also underlie the decision to methylate the box in the first place.In other words,de novo methylation may not occur once at a discrete and perhaps rather in-accessible stage of germ-cell development,but may hap-pen repeatedly(assisted by DNMT1)as embryonic cells divide.

Unusual DNA structures and RNAi as triggers

for de novo methylation

Studies of purified DNMT1revealed that the enzyme prefers to methylate unusual DNA structures in vitro (Smith et al.1991;Laayoun and Smith1995).This led to the idea that such structures might be generated during recombination between repetitive elements or during transposition events and directly trigger de novo meth-ylation(Bestor and Tycko1996).Subsequent evidence, however,does not support a role for DNMT1in de novo methylation in vivo(Lyko et al.1999;Howell et al. 2001),and therefore the biological significance of its predilection for deformed DNA is uncertain.There is evidence for transfer of methylation from one copy of a sequence to a second previously unmethylated copy of the same sequence in the fungus Ascobolus(Colot et al. 1996).The process might use mechanisms involved in homologous DNA recombination and may therefore in-volve deformation of DNA.How identical sequences sense one another and transfer epigenetic information remains unknown,however.

Exciting recent developments in the DNA methyl-ation field have arisen through molecular genetic studies of posttranscriptional gene silencing in plants.Double-stranded RNA directs the destruction of transcripts con-taining the same sequence,but there is compelling evi-dence that it can also direct de novo methylation of ho-mologous genomic DNA(Wassenegger et al.1994; Bender2001;Matzke et al.2001).Posttranscriptional gene silencing by double-stranded RNA is probably an ancient genome defence system because it occurs in fungi,plants,and animals;but DNA methylation is not an obligatory accompaniment,as silencing is efficient in C.elegans in the complete absence of genomic m5C. Even in the fungus Neurospora,where transgene arrays are often methylated,DNA methylation is not required for posttranscriptional gene silencing(or quelling;Co-goni et al.1996).There are also specific features of RNA-directed DNA methylation that may not occur in ani-mals;notably the occurrence of methylation at multiple non-CpG cytosines in an affected DNA sequence tract. Although there is evidence for non-CpG methylation in ES cells,most probably owing to DNMT3A,which strongly methylates CpA as well as CpG(Ramsahoye et al.2000;Gowher and Jeltsch2001),non-CpG methyl-ation is barely detectable in adult cells(Ramsahoye et al. 2000).Plants have a CpG methylation system,but it does not appear to be essential for RNA-directed gene silencing(for reviews,see Wassenegger et al.1994; Bender2001;Matzke et al.2001).Optimism that RNA-directed de novo methylation will also apply in mam-mals is tempered by this sequence disparity,and by the absence so far of a clear demonstration that mammalian double-stranded RNA leads to DNA methylation-medi-ated gene silencing.

Transcriptionally silent chromatin as a de novo methylation target

Several lines of evidence suggest that DNA methylation does not intervene to silence active promoters,but af-fects genes that are already silent.It was reported many years ago that retroviral transcription is repressed in em-bryonic cells at～2d after infection,whereas de novo methylation is delayed until～15d(Gautsch and Wilson 1983;Niwa et al.1983).De novo methylation of proviral sequences in embryo cells depends on DNMT3A and DNMT3B(Okano et al.1999),but initial retroviral shut-down occurs as usual even when both these de novo methyltransferases are absent(Pannell et al.2000). Clearly,de novo methylation is not required for silenc-ing in the first instance,reinforcing the view that meth-ylation is a secondary event.

Methylation of genes that are already silent is also observed during X chromosome inactivation in the mammalian embryo.Kinetic studies showed that the phosphoglycerate kinase gene is silent on the mamma-lian inactive X chromosome before methylation of its CpG-island promoters occurs(Lock et al.1987).Subse-quent studies of the mouse,in which the process is best understood,have established that expression of a non-coding chromosomal RNA from the Xist gene on the inactive X chromosome triggers the inactivation process

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GENES&DEVELOPMENT9

in cis.Specifically,activation of the Xist gene and onset of its late replication precede CpG-island methylation by several days(Keohane et al.1996;Wutz and Jaenisch 2000).In other words,methylation affects the X chromo-some on which genes are already shut down by other mechanisms.Is transcriptional inertia during embryo-genesis the trigger for de novo methylation?Studies of the origin of methylation-free CpG islands offer some support for this idea.The coincidence between CpG is-lands and promoters is striking(Bird1987),and foot-printing shows that the5?extremity of CpG islands of-ten corresponds to the region occupied by transcription factors in vivo(Cuadrado et al.2001).Even when CpG islands are identified in unusual locations,they have turned out to correspond to promoters.For example,a CpG island located in intron2of the Igf2r gene is an active promoter(Wutz et al.1997;Lyle et al.2000),as is a CpG island that covers exon2of the class II major histocompatibility gene(MacLeod et al.1998).The po-tential importance of promoter function in the genesis of CpG islands is highlighted by studies in transgenic mice. CpG-island-containing transgenes normally faithful re-produce their methylation-free character,but their im-munity to methylation is lost if promoter function is impaired(Brandeis et al.1994;MacLeod et al.1994). Similarly,viral DNA integrated into ES cell genomes by homologous recombination becomes methylated when the promoter is weakened by absence of an enhancer,but excludes methylation when an enhancer is present (Hertz et al.1999).A parsimonious interpretation of the results is that failure to transcribe invites de novo meth-ylation(see Fig.2below),although other potential ex-planations(Brandeis et al.1994;Mummaneni et al.1998) cannot be discounted.

The signal for this putative gene silence-related de novo methylation is unknown,but the possibility that chromatin states inform the DNA methylation machin-ery is attractive(Selker1990).The acetylation and methylation state of nucleosomal histones is tightly cor-related with transcriptional activity(Jenuwein and Allis2001)and could be read by the methylation ma-chinery,leading it to either methylate or fail to methyl-ate a particular domain.Indeed,recent work on Neuros-pora(Tamaru and Selker2001)has shown an intimate link between histone methylation and DNA methyl-ation in that fungus,as mutation of a histone methyl-transferase that methylates Lys9of histone H3abol-ished genomic methylation.In mammalian and yeast systems,histone H3Lys9methylation is associated with transcriptionally repressed heterochromatin(Ban-nister et al.2001;Nakayama et al.2001;Noma et al. 2001;Zhang and Reinberg2001).If the dependence of DNA methylation on prior histone methylation turns out to be applicable to mammals,this would further strengthen the argument that DNA methylation is tar-geted to genes that are already silent.The nature of the molecular cues that trigger transfer of methyl groups to unmethylated DNA should be illuminated by ongoing studies of multiprotein complexes that contain DNA methyltransferases(Fuks et al.2000,2001;Robertson et al.2000;Bachman et al.2001)and the identification of genes that modify DNA methylation patterns(Weng et al.1995).

Consequences of methylation gain:stable transcriptional silencing of genes

Why methylate genes that are already silent?A plausible answer is:to silence them irrevocably.Methylation clearly contributes to the stability of inactivation,be-cause both X inactivation(Mohandas et al.1981a; Graves1982;Venolia et al.1982)and retroviral silencing (Stewart et al.1982;Jaenisch et al.1985)can be relieved by treatment of somatic cells with demethylating agents.Individuals who lack DNMT3B show reduced methylation of some CpG islands on the inactive X chro-mosome and also silence X-linked genes imperfectly (Miniou et al.1994;Hansen et al.2000).The implication that irreversibility involves DNA methylation is sup-ported by the frequent reactivation of an X-linked trans-gene in mouse embryo cells and in cultured somatic cells when DNMT1is absent or inhibited(Sado et al.2000). This view is sustained by differences in the stability of inactivity states pre-and postmethylation.For example, X inactivation caused by expression of an Xist transgene in embryonic stem cells is initially reversed when the Xist gene is shut down,but after3d,inactivation be-comes irreversible and independent of Xist(Wutz and Jaenisch2000).Irreversibility may reflect the arrival of promoter methylation.

In artificial systems,DNA methylation represses tran-scription in a manner that depends on the location and density of the methyl-CpGs relative to the promoter (Boyes and Bird1992;Hsieh1994;Kass et al.1997a,b). But what genes are affected by DNA methylation-medi-ated gene silencing?Early studies relied on the use of the demethylating drug5-azacytidine(Jones and Taylor 1980),which was shown to activate genes on the inac-tive X in rodent–human cell hybrids(Mohandas et al. 1981b;Graves1982).More recently,mice and murine cell lines lacking DNMT1(Li et al.1992)have clarified the effects of DNA methylation on gene expression.In placental mammals,repression of X-linked genes fol-lows expression of Xist,which sets in train the inactiva-tion process,culminating in widespread methylation of CpG islands.The active X chromosome,on the other hand,must be protected from silencing,and this requires repression of Xist and again depends on methylation (Panning and Jaenisch1996).An intact DNA methyl-ation system is also essential for genomic imprinting, because deletion of Dnmt1leads to disruption of the monoallelic expression of several imprinted genes(Li et al.1993).

Both X inactivation and genomic imprinting involve silencing of one allele only,leaving the other unaffected. An unusual set of genes that are active in the germ line, most of which are X-linked,appears to use methylation for complete silencing in somatic cells(De Smet et al. 1996,1999).Several of the human and murine MAGE genes,for example,have CpG-island promoters that are

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methylation-free in germ cells,but are methylated in somatic cells of the adult.The genes were discovered as novel antigens in tumors,where genomic methylation levels are often low and MAGE-gene CpG islands are undermethylated.MAGE expression can be induced by treating nonexpressing cells with demethylating agents, supporting the idea that methylation is an important component of the repression of these genes in somatic cells.

Transposable element silencing as a consequence

of DNA methylation

Another well-documented consequence of DNA meth-ylation deficiency is the activation of transposable ele-ment-derived promoters.Like much of the mammalian genome,transposable element-related sequences are heavily methylated and transcriptionally silent in so-matic cells.Mouse cells,for example,normally repress transcription of intracisternal A particle(IAP)elements, which constitute a homogeneous and transpositionally active family of elements.In embryos lacking DNMT1, transcription of IAP elements is massively induced,ar-guing that methylation is normally responsible for their repression(Walsh et al.1998).Derepression of LINE (Woodcock et al.1997)and SINE(Liu et al.1994)pro-moters in the human genome also occurs when DNA methylation is reduced.The most abundant SINE in the human genome is the Alu family,which consists of sev-eral hundred thousand elements(Smit1999).Only a tiny minority of elements are capable of transposition(<1%), but many carry functional promoters.Interestingly, these promoters can be activated by stress of various kinds without altering DNA methylation(Liu et al. 1995;Chu et al.1998),although artificial demethylation also stimulates expression.

The biological significance of transposable-element re-pression is uncertain.Two kinds of explanation have been discussed:either that repression is required to pre-vent DNA damage due to unconstrained transposition (the genome defence model;Yoder et al.1997);or that transcription of a large excess of irrelevant promoters would constitute an unacceptable level of transcrip-tional noise that would interfere with gene expression programs(Bird1995).Increased transcription of elements in human and mouse cells has not so far been found to lead to increased transposition.In undermethylated can-cer cells that show transposon promoter activity,for ex-ample,mutations caused by transposition are exceed-ingly rare.It has,however,been claimed that rampant transposition and reduced methylation are linked in the case of an interspecific hybrid marsupial(Waugh O’Neill et al.1998).The hybrid wallaby concerned was found to contain an abundant transposable element near the cen-tromeres of one parental chromosome set,but not the other.Surprisingly,this element could not be detected in either of the presumed parent species,and was therefore hypothesized to have been assembled from related frag-ments in the parental genomes following fertilization.It was suggested that,because of perceived depression of methylation levels in the hybrid embryo,the emergent element became transpositionally hyperactive,being tar-geted exclusively to one parental genome.The parents of the hybrid were not available to verify this unprec-edented scenario.

Phylogenetic studies of genomic methylation patterns in animals have not yet offered support for the genome defence model.Effective silencing due to sequence rep-etition has been observed in Drosophila and C.elegans, but it is associated with the polycomb group of proteins or posttranscriptional gene silencing(Birchler et al. 2000).The possibility that the low level of m5C in Dro-sophila(Lyko et al.2000)is relevant to silencing has not yet been addressed.Studies of the sea squirt C.intesti-nalis,a chordate belonging to the same phylum as ver-tebrates,but which does not exhibit global methylation of the genome,revealed that genes were often present in domains of methylated DNA,whereas transposable ele-ment families,some of which appeared to be mobile in the population,were unmethylated(Simmen et al.1999). This is the opposite of expectation,but may represent a frequent situation in invertebrates,which account for >95%of animal species(Tweedie et al.1997). Colonization of the genome by transposable elements can only occur in the germ-cell lineage because somatic transposition events leave no heritable trace.Paradoxi-cally,transposable elements are often transcriptionally active and unmethylated in germ cells and totipotent ES cells(for review,see Bird1997).IAP elements,for ex-ample,become unmethylated during the gonial prolif-eration phase,when primordial germ cell number in-creases from～75to～25,000(Walsh et al.1998).The fre-quent absence of DNA methylation in germ cells,when transposition can do long-term damage(Malik et al. 1999),contrasts with its repressive presence in somatic cells,where transposition would be an evolutionary dead end.It is too early to discount the possibility that trans-poson promoters,most of which belong to degenerate elements that are incapable of transposition,must be silenced to suppress transcriptional noise. Mechanisms of DNA methylation-mediated transcriptional repression

Why does DNA methylation interfere with transcrip-tion?Two modes of repression can be envisaged,and it is likely that both are biologically relevant.The first mode involves direct interference of the methyl group in bind-ing of a protein to its cognate DNA sequence(Fig.1). Many factors are known to bind CpG-containing se-quences,and some of these fail to bind when the CpG is methylated.Strong evidence for involvement of this mechanism in gene regulation comes from studies of the role of the CTCF protein in imprinting at the H19/Igf2 locus in mice(Bell and Felsenfeld2000;Hark et al.2000; Szabo et al.2000;Holmgren et al.2001).CTCF is asso-ciated with transcriptional domain boundaries(Bell et al. 1999)and can insulate a promoter from the influence of remote enhancers.The maternally derived copy of the Igf2gene is silent owing to the binding of CTCF between

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its promoter and a downstream enhancer.At the pater-nal locus,however,these CpG-rich binding sites are methylated,preventing CTCF binding and thereby al-lowing the downstream enhancer to activate Igf2expres-sion.Although there is evidence that H19/Igf2imprint-ing involves additional processes (Ferguson-Smith and Surani 2001),the role of CTCF represents one of the clearest examples of transcriptional regulation by DNA methylation.

The second mode of repression is opposite to the first,as it involves proteins that are attracted to,rather than repelled by,methyl-CpG (Fig.1).A family of five methyl-CpG-binding proteins has been characterized that each contains a region closely related to the methyl-CpG-binding domain (MBD)of MeCP2(Nan et al.1993,1997;Cross et al.1997;Hendrich and Bird 1998).Four of these proteins —MBD1,MBD2,MBD3,and MeCP2—have been implicated in methylation-dependent repression of transcription (for review,see Bird and Wolffe 1999).An unrelated protein Kaiso has also recently been shown to bind methylated DNA and bring about methylation-de-pendent repression in model systems (Prokhortchouk et al.2001).In vitro,Kaiso requires a 5?m 5CGm 5CG motif,and binding is highly dependent on the presence of meth-ylation.The presence of multiple methyl-CpG-binding proteins with repressive properties supports the argu-ment that these may be important mediators of the methylation signal,but their involvement in specific processes that require transduction of the DNA methyl-ation signal has yet to be shown.Targeted mutation of the gene for MeCP2is,however,associated with neuro-logical dysfunction in humans and mice (Amir et al.1999;Chen et al.2001;Guy et al.2001),and mutation of the mouse Mbd2gene leads to a maternal behavior de-fect (Hendrich et al.2001).

Excluding DNA methylation by denying access The preceding discussion has considered some mecha-nistic aspects of de novo DNA methylation and its bio-logical consequences.Although methylation affects most of the mammalian genome,it is conspicuously ab-sent from certain regions.Ways in which these non-methylated domains may arise will now be considered.A simple mechanism for creating a nonmethylated do-main within an otherwise densely methylated genome is to mask a stretch of DNA by protein binding.The DNA-binding protein would accomplish this passive demeth-ylation by,for example,sterically excluding DNMTs (Bird 1986).The feasibility of this mechanism has been verified using an artificially methylated episome con-taining EBNA1or lac repressor binding sites (Hsieh 1999a;Lin et al.2000).The idea that CpG islands are entirely attributable to exclusion of this kind is in doubt,however,as in vivo footprinting and nuclease accessibil-ity studies show CpG islands to be more accessible to proteins (nucleases)than bulk genomic DNA,not less (Tazi and Bird 1990).Of course,it is possible that pro-tection is only present at the transient embryonic stage when mammalian de novo methylation occurs and has therefore escaped detection.A protein that is reported to bind unmethylated CpGs might be a candidate CpG-is-land protector (Voo et al.2000).

Immunity to DNA methylation caused by transcriptionally active chromatin:the origin of unmethylated CpG islands

Many of the known biological effects of DNA methyl-ation are associated with CpG islands.It has been argued above that their methylation in the early embryo

follows

Figure 1.Mechanisms of transcriptional repression by DNA methylation.A stretch of nucleosomal DNA is shown with all CpGs methylated (red circles).Below the diagram is a transcription factor that is un-able to bind its recognition site when a methylated CpG is within it.Many tran-scription factors are repelled by methyl-ation,including the boundary element protein CTCF (see text).Above the line are protein complexes that can be attracted by methylation,including the methyl-CpG-binding protein MeCP2(plus the Sin3A histone deacetylase complex),the MeCP1complex comprising MBD2plus the NuRD corepressor complex,and the un-characterized MBD1and Kaiso complexes.MeCP2and MBD1are chromosome-bound proteins,whereas MeCP1may be less tightly bound.Kaiso has not yet been shown to associate with methylated sites in vivo.

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12GENES &DEVELOPMENT

silencing events that are likely to be DNA methylation-independent.If transcriptional silence indeed triggers DNA methylation,then the corollary is that promoter activity early in development should create a methyl-ation-free CpG island (Fig.2).In other words,unmethyl-ated CpG islands might be footprints of embryonic pro-moter activity.An obvious prediction of this model is that all unmethylated CpG islands,including those at promoters of highly tissue-specifically expressed genes,should contain promoters that function during early de-velopment when the methylation memory system is most active.Although very limited,the data so far favor this theory,because a CpG-island promoter whose prod-uct RNA is not expected to occur in the early embryo (?-globin)is nevertheless expressed,whereas transcripts from a CpG-deficient promoter (?-globin)are not de-tected (Daniels et al.1997).Similarly,expression of the 68k neurofilament gene,which has a CpG-island pro-moter,was detected in ES cells,but opsin and casein genes,which are CpG-deficient genes,appeared to be silent (MacLeod et al.1998).

Why should active promoter regions escape de novo methylation?CpG islands often colocalize with origins of DNA replication (Delgado et al.1998),and,according to one speculation,an early replication intermediate cre-ates the DNA methylation-free footprint (Antequera and Bird 1999).A more direct (but not mutually exclusive)mechanism would involve the sensing of chromatin states by the de novo methylation system as discussed above.Whereas histone H3tails modified by methyl-ation on Lys 9might recruit DNA methyltransferases (Tamaru and Selker 2001),modifications associated with active chromatin,such as acetylation of H3or H4or methylation of Lys 4of histone H3,may actively exclude these enzymes.Biochemical evidence addressing this is-sue is eagerly awaited.Active demethylation of DNA

Protection against de novo methylation by bound pro-teins or chromatin can ensure that DNA methylation never reaches a DNA sequence domain.

Unmethylated

Figure 2.A hypothetical scenario relating embryonic transcriptional activity to DNA methylation status in mammals.Starting from a notional transcription ground state,embryonic demethylation leads to substitution of methylated sites (red circles)by nonmethyl-ated sites (yellow circles).Two alternative fates are then envisaged:either transcription persists leading to restoration of the unmeth-ylated CpG island (bracket)flanked by methylated non-island-flanking DNA (pink arrows);or transcription is extinguished by other mechanisms in the embryo and this invites de novo methylation of the CpG island and its flanks.In this way the activity of embryonic promoters is imprinted for the duration of that somatic lifetime.

DNA methylation and epigenetic memory

GENES &DEVELOPMENT 13

domains could also arise by actively removing the modi-fication from DNA.This so-called active demethylation could be accomplished either by the thermodynamically unfavorable breakage of the carbon—carbon bond that links the pyrimidine to its methyl group,or by a repair-like process that excises the m5C base or nucleoside, leading to its replacement with C(Kress et al.2001). Several laboratories have striven to isolate demethylase enzymes(for review,see Wolffe et al.1999).The most impressive catalytic activity was shown by a fraction derived from human cells(Ramchandani et al.1999)that was subsequently identified as MBD2(Bhattacharya et al.1999).The expressed protein reportedly showed ro-bust demethylation in vitro in the absence of added co-factors and released methanol as a by-product.Attempts to observe this property of MBD2in other laboratories have not been successful.

A cell extract showing demethylase activity was de-tected in rat myoblast cells(Weiss et al.1996).Initial indications that the reaction was RNA-dependent were not sustained upon further enrichment of the activity (Swisher et al.1998).An RNA-containing demethylating complex was,however,reported in chicken cells(Jost et al.1997,1999).These investigators searched for proteins with m5C-DNA glycosylase activity and identified the previously known thymine DNA glycosylase TDG, which can remove the pyrimidine base from T:G or U:G mismatches(Zhu et al.2000b).MBD4,an unrelated DNA glycosylase with similar properties,was also found to be active against m5C:G pairs(Zhu et al.2000a).As the efficiency of these reactions was much lower than that seen with the cognate mismatched substrates,it might be argued that the m5C glycosylase activity rep-resents a minor side reaction of little in vivo signifi-cance.Set against this is evidence that stable expression of a chicken TDG results in significant activation and concomitant demethylation of a reporter gene driven by a methylated ecdysone-retinoic acid-responsive pro-moter(Zhu et al.2001).The normally silent reporter could also be activated by demethylation with5-azacyti-dine,but generalized demethylation of the genome was not observed in TDG transfected cells.Previous studies showed an association between retinoid receptors and TDG,and implicated TDG in transcriptional activation (Um et al.1998).Time will tell if the stimulation of retinoid-responsive promoters by TDG depends on its demethylating activity.

The need to isolate demethylating enzymes has be-come more acute with the finding that the paternal ge-nome is subject to active demethylation soon after fer-tilization(Mayer et al.2000;Oswald et al.2000).Similar processes have been reported in pig and bovine embryos (Bourc’his et al.2001;Kang et al.2001a,b).This dramatic illustration of methylation loss in the absence of DNA replication raises questions about the prevalence of de-methylation by this mechanism.Interestingly,the ma-ternal genome,which also demethylates during early mouse development,does so by a different mechanism: passive failure to methylate progeny stands(Rougier et al.1998).Why should maternal and paternal genomes choose such different routes to the same end?An intrigu-ing possibility is that the parental struggle over maternal resources for the embryo that is thought to underlie ge-nomic imprinting(Moore and Haig1991)may be in-volved.The oocyte may be equipped to directly disarm the sperm genome of methylation imprints that might overexploit maternal resources(Reik and Walter2001).It is even possible that the paternal genome,in delayed retaliation,may organize a campaign of interference with the maintenance methylation(e.g.,by exporting maternal DNMTs to the cytoplasm).The extraordinary need for an oocyte variant of DNMT1to translocate into the nucleus for only one cleavage cycle(the doubling from8to16cells;Howell et al.2001)could represent maternal measures to compensate for interference of this kind.

Consequences of methylation loss:gene activation during development

Interest in DNA methylation has long been fueled by the notion that strategic loss of methyl groups during devel-opment could lead to activation of specific genes in the appropriate lineage.As has been emphasized(Walsh and Bestor1999),much of the evidence for this scenario is inconclusive,but recent studies have revived the idea.In the frog,gene expression is suppressed from fertilization until the mid-blastula stage(～5000cells),at which time transcription is activated.Inhibition of DNMT1using an antisense strategy caused reduced methylation and pre-mature activation of certain genes,suggesting a direct role for DNA methylation in maintaining their early si-lence prior to the blastula stage(Stancheva and Meehan 2000).Deletion of the Dnmt1gene in cultured somatic cells of the mouse also caused widespread gene activa-tion(Jackson-Grusby et al.2001).About10%of all genes detected using microarray technology were activated, whereas only1%–2%were down-regulated.Some of the up-regulated genes are normally only expressed in termi-nally differentiated cells.These findings raise the possi-bility that DNA methylation contributes to silencing of tissue-specific genes in nonexpressing cells,and they confirm DNA methylation as a global repressor of gene expression.The scenario has been modeled using an ar-tificial construct that contained a DNA sequence ca-pable of excluding methylation locally during early de-velopment(Siegfried et al.1999).When this sequence was present,the reporter gene stayed unmethylated dur-ing development,and widespread expression occurred. Deletion of the element in situ in the early embryo led to methylation of the reporter gene and concomitant si-lencing in several adult tissues.

A subtle potential role for loss of methylation at a specific gene has been reported for the rat tyrosine ami-notransferase gene(Thomassin et al.2001).When a methylated form of this gene is induced by glucocorti-coids,delayed demethylation occurs at specific sites in an enhancer and additional DNA-associated factors are subsequently recruited.Demethylation(whether active or passive is not known)persists after the wave of TAT

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14GENES&DEVELOPMENT

expression has subsided,and reinduction of the silent gene by a further hormone treatment is significantly stronger as a result.This system provides a model for a DNA methylation-mediated memory of the first hor-mone induction(Kress et al.2001).Its significance is somewhat less certain in normal development,however, because demethylation of these sites occurs before the gene becomes hormone-inducible.

There is suggestive evidence that programmed rear-rangement of the immunoglobulin genes during B-cell development may involve DNA methylation(Mosto-slavsky et al.1998).Demethylation of one of the two parentally derived alleles of the kappa light chain gene is observed in small pre-B cells,and there is evidence that this early loss of methylation predisposes the affected allele to rearrangement.By precluding rearrangement of one allele,differential DNA methylation may help to explain allelic exclusion at the kappa chain locus.It is not certain whether transcriptional regulation per se plays a role,although the process is dependent on the intronic and3?kappa gene enhancers.

Loss of genome integrity as a consequence of DNA methylation loss?

Early studies with the DNA-methylation inhibitor 5-azacytidine revealed bizarre chromosomal rearrange-ments in treated cultured cells(Viegas-Pequignot and Dutrillaux1976).Although these findings might be at-tributed to the effects of reduced DNA methylation,they could also be a result of the chemical reactivity of the incorporated base analog,in particular,its ability to cross-link proteins to DNA(Juttermann et al.1994).The former possibility is supported somewhat by the finding that mitogen-stimulated lymphocytes from patients with mutations in DNMT3B show very similar chromo-some rearrangements,involving coalescence of centro-meric regions that contain methylation-deficient repeti-tive sequences(Jeanpierre et al.1993;Xu et al.1999). Oddly,the rearrangements are not seen in cells of the patients,despite similar hypomethylation of these re-gions.It seems that loss of genomic integrity is not an obligatory consequence of hypomethylation of juxtacen-tromeric repeat elements.

At a finer level,two laboratories have examined the effects of greatly reduced DNA methylation levels on mutation rates in mouse embryonic stem cells,with somewhat differing results.In one study,the mutation rate at two endogenous loci was found to have increased ～10-fold compared to the same loci in wild-type cells (Chen et al.1998),suggesting that lack of methylation predisposed to aberrant recombination events.A second study examined transgenes of exogenous origin using a selection system to detect mutations(Chan et al.2001). This allowed screening of large numbers of mutations at two independent loci,but neither point mutations nor genomic rearrangements were increased under condi-tions of limiting DNA methylation.In fact,mutations appeared to be suppressed by genomic hypomethylation. These inconsistencies raise questions about the pro-posed relationship between genome integrity and DNA methylation that will need to be addressed by further research.

Developmental memory:DNA methylation

and Polycomb/trithorax complexes

as interchangeable systems

The foregoing discussion has highlighted features of the DNA methylation system in mammals that resemble another established system of cellular memory:Pc-G/ trx.The final section of the review will compare the two systems.The credentials of Pc-G/trx protein complexes as an epigenetic system in development are compelling (Paro et al.1998;Pirrotta1999;Francis and Kingston 2001).This multiprotein assembly is targeted to specific regions of the genome where it effectively freezes the embryonic expression status of a gene,be it active or inactive,and propagates that state stably through devel-opment.Elegant experiments with model gene con-structs have shown that brief activation(or inactivation) of a promoter during early Drosophila development leads to stable activity(or inactivity)thereafter(Cavalli and Paro1998,1999;Poux et al.2001).Attempts to alter expression at most other stages of development were un-successful,indicating that there is a window of time dur-ing which transcription patterns can be committed to developmental memory.The Pc-G/trx system is reactive rather than proactive,as the setting up of segment-spe-cific patterns of active genes is not disrupted by muta-tions in Pc-G group genes.Only the capacity to sustain the patterns is lost in the mutants.This ability to copy and propagate the expression patterns without influenc-ing or perturbing them makes this a subtle and flexible memory system.Little is known,however,about the mechanisms responsible for the heritability of Pc-G/trx. What do Pc-G/trx and DNA methylation in mammals have in common?First,both systems are able to repress transcription in a heritable manner.Second,both appear to be reactive in that they lock in expression states that they played no part in setting up(e.g.,DNA methylation in viral genome silencing and CpG-island methylation on the X chromosome).Third,both are activated prima-rily during a discrete window of time in early develop-ment.Thus,like Pc-G/trx,DNA methylation has the properties of a developmental memory.

What is memorized by DNA methylation?Arguably, its major role is to stably demarkate by its absence a set of embryonically active promoters,namely,CpG is-lands,so that they remain potentially active throughout development and adulthood.At the same time,regions devoid of promoter activity in the embryo become meth-ylated and carry this repressive influence with them through development.The degree of repression may be weak or strong depending on the density of methylation (Boyes and Bird1992;Hsieh1994).Thus,CpG islands that are silenced by other mechanisms during embryo-genesis would acquire dense methylation leading to ir-reversible silencing.When,however,the density of methylated CpGs is low,as it is in most of the genome,

DNA methylation and epigenetic memory

GENES&DEVELOPMENT15

repression is likely to be weak and may be overcome by the presence of strong activators.Weak repression of tis-sue-specific genes(e.g.,?-globin)that are embedded in regions of low-density methylation may contribute to their silence in inappropriate tissues.

It is proposed here that DNA methylation and Pc-G/ trx are alternative systems of cellular memory that are interchangeable over evolutionary time.In C.elegans and Drosophila,for example,Pc-G group proteins (Birchler et al.2000)have been implicated in silencing of repetitive-element transcription in somatic cells, whereas DNA methylation may play this role in mam-mals(Yoder et al.1997).The involvement of DNA meth-ylation in genome defence may,therefore,be to memo-rize the silent state of elements imposed by primary ge-nome defence systems.The most striking evidence for interchangeability is the finding that X chromosome in-activation in extraembryonic tissues of the mouse de-pends on the polycomb group protein Eed.Loss of the eed gene leads to reactivation of the inactive X in extra-embryonic tissue,but has no effect in somatic cell types (Wang et al.2001).In contrast,Dnmt1mutations reac-tivate the inactive X of the embryo proper,but not the extraembryonic inactive X(Sado et al.2000).The finding that certain CpG islands on the inactive X chromosome are methylated in somatic cells but not in extraembry-onic tissues(Iida et al.1994)fits with the view that methylation replaces Pc-G in somatic tissues.Therefore, even within a single species,it appears that different tissues employ Pc-G/trx and DNA methylation inter-changeably.From an evolutionary perspective,it is pos-sible that varying degrees of functional substitution by Pc-G(or vice versa)can explain the dramatic differences between DNA methylation levels across animal species. Concluding remarks

Our understanding of the relationship between DNA methylation and transcriptional control is growing fast, but is still far from complete.Ongoing biochemical analysis of the growing number of components of the DNA methylation system(and their partners),coupled with genetic approaches,will strengthen the links be-tween DNA methylation and mainstream transcrip-tional mechanisms.Regulation of gene expression is complex(Lemon and Tjian2000),and the emerging evi-dence hints that the roles of DNA methylation will be too.It may be unrealistic to expect that any unified theory will encompass all the biological consequences of DNA methylation.

Least understood are the mechanisms by which meth-ylation patterns are generated.Following consideration of the criteria for attracting and repelling DNA methyl-ation,this review has entertained the possibility that a primary function of de novo DNA methylation is to memorize patterns of embryonic gene activity,creating CpG islands that are competent for transcription throughout development,or their antithesis,regions that are methylated and transcriptionally incompetent. The idea depends on evidence that methylation does not intervene to silence genes that are actively transcribed, but only affects genes that have already been shut down by other means.There is reason to believe that transcrip-tional activity may somehow imprint the methylation-free status of CpG islands.The involvement of DNA methylation in inactivation of transposable elements could likewise be due to its capacity for stabilizing the transcriptional shutdown organized by other systems. Parallels between these emerging attributes of DNA methylation and the Pc-G system in Drosophila suggest that both are mechanisms for sensing and propagating cellular memory.

Acknowledgments

I am grateful to Eric Selker,Bernard Ramsahoye,Helle J?r-gensen,Brian Hendrich,and Catherine Millar for comments on the manuscript.Research by A.B.is supported by The Wellcome Trust.

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GENES&DEVELOPMENT21

半导体材料课程教学大纲

半导体材料课程教学大纲 一、课程说明 （一）课程名称：半导体材料 所属专业：微电子科学与工程 课程性质：专业限选 学分： 3 （二）课程简介：本课程重点介绍第一代和第二代半导体材料硅、锗、砷化镓等的制备基本原理、制备工艺和材料特性，介绍第三代半导体材料氮化镓、碳化硅及其他半导体材料的性质及制备方法。 目标与任务：使学生掌握主要半导体材料的性质以及制备方法，了解半导体材料最新发展情况、为将来从事半导体材料科学、半导体器件制备等打下基础。 （三）先修课程要求：《固体物理学》、《半导体物理学》、《热力学统计物理》； 本课程中介绍半导体材料性质方面需要《固体物理学》、《半导体物理学》中晶体结构、能带理论等章节作为基础。同时介绍材料生长方面知识时需要《热力学统计物理》中关于自由能等方面的知识。 （四）教材：杨树人《半导体材料》 主要参考书：褚君浩、张玉龙《半导体材料技术》 陆大成《金属有机化合物气相外延基础及应用》 二、课程内容与安排 第一章半导体材料概述 第一节半导体材料发展历程 第二节半导体材料分类 第三节半导体材料制备方法综述 第二章硅和锗的制备 第一节硅和锗的物理化学性质 第二节高纯硅的制备 第三节锗的富集与提纯

第三章区熔提纯 第一节分凝现象与分凝系数 第二节区熔原理 第三节锗的区熔提纯 第四章晶体生长 第一节晶体生长理论基础 第二节熔体的晶体生长 第三节硅、锗单晶生长 第五章硅、锗晶体中的杂质和缺陷 第一节硅、锗晶体中杂质的性质 第二节硅、锗晶体的掺杂 第三节硅、锗单晶的位错 第四节硅单晶中的微缺陷 第六章硅外延生长 第一节硅的气相外延生长 第二节硅外延生长的缺陷及电阻率控制 第三节硅的异质外延 第七章化合物半导体的外延生长 第一节气相外延生长（VPE） 第二节金属有机物化学气相外延生长（MOCVD） 第三节分子束外延生长（MBE） 第四节其他外延生长技术 第八章化合物半导体材料（一）：第二代半导体材料 第一节 GaAs、InP等III-V族化合物半导体材料的特性第二节 GaAs单晶的制备及应用 第三节 GaAs单晶中杂质控制及掺杂 第四节 InP、GaP等的制备及应用 第九章化合物半导体材料（二）：第三代半导体材料 第一节氮化物半导体材料特性及应用 第二节氮化物半导体材料的外延生长 第三节碳化硅材料的特性及应用 第十章其他半导体材料

吸波材料知识介绍系列

吸波材料知识介绍系列—————之一 吸波材料简介 在解决高频电磁干扰问题上，完全采用屏蔽的解决方式越来越不能满足要求了。因为诸多设备中，端口的设置及通风、视窗等的需求使得实际的屏蔽措施不可能形成像法拉第电笼那样的全屏蔽电笼，端口尺寸问题是设备高频化的一大威胁。另外，困扰人们的还有另外一个问题，在设备实施了有效的屏蔽后，对外干扰问题虽然解决了，但电磁波干扰问题在屏蔽系统内部仍然存在，甚至因为屏蔽导致干扰加剧，甚至引发设备不能正常工作。这些都是屏蔽存在的问题，也正是因为这些问题的存在，吸波材料有了用武之地。 吸波材料是指能够有效吸收入射电磁波并使其散射衰减的一类材料，它通过材料的各种不同的损耗机制将入射电磁波转化成热能或者是其它能量形式而达到吸收电磁波目的。不同于屏蔽解决方案，其功效性在于减少干扰电磁波的数量。既可以单独使用吸收电磁波，也可以和屏蔽体系配合，提高设备高频功效。 目前常用的吸波材料可以对付的电磁干扰频段范围从0.72GHz到40GHz。当然应用在更高和更低频段上的吸波材料也是有的。吸波材料大体可以分成涂层型、板材型和结构型；从吸波机理上可以分成电吸收型、磁吸收型；从结构上可以分为吸收型、干涉型和谐振型等吸波结构。吸波材料的吸波效果是由介质内部各种电磁机制来决定，如电介质的德拜弛豫、共振吸收、界面弛豫磁介质畴壁的共振弛豫、电子扩散和微涡流等。 吸波材料的损耗机制大致可以分为以下几类：其一，电阻型损耗，此类吸收机制与材料的导电率有关的电阻性损耗，即导电率越大，载流子引起的宏观电流（包括电场变化引起的电流以及磁场变化引起的涡流）越大，从而有利于电磁能转化成为热能。其二，电介质损耗，它是一类与电极有关的介质损耗吸收机制，即通过介质反复极化产生的“摩擦”作用将电磁能转化成热能耗散掉。电介质极化过程包括：电子云位移极化，极性介质电矩转向极化，电铁体电畴转向极化以及壁位移等。其三，磁损耗，此类吸收机制是一类与铁磁性介质的动态磁化过程有关的磁损耗，此类损耗可以细化为：磁滞损耗，旋磁涡流、阻尼损耗以及磁后效效应等，其主要来源是与磁滞机制相似的磁畴转向、磁畴壁位移以及磁畴自然共振等。此外，最新的纳米材料微波损耗机制是目前吸波材料研究的一大热点。由于篇幅所限，本文对吸波材料的损耗机制仅做了最为简约的叙述，对其详述及其结构设计及结构对吸波效能的影响等方面将在以后的文章中做出解释。 总之，高速发展的新科技正引领着世界范围内的各行各类电气、电子设备向高频化、小型化方向发展，高频EMI问题必将越发突显，吸波材料必然有越来越广阔的应用空间。

(能源化工行业)常用化工原料

（能源化工行业）常用化工 原料

硫酸镍 化学式及产品介绍 化学式为NiSO4 硫酸镍分为有无水物、六水物和七水物三种。商品多为六水物，有α-型和β-型俩种变体，前者为蓝色四方结晶，后者为绿色单斜结晶。溶于水，水溶液呈酸性，易溶于醇和氨水。 二、作用和用途 硫酸镍主要用于电镀工业，是电镀镍和化学镍的主要镍盐，也是金属镍离子的来源，能在电镀过程中，离解镍离子和硫酸根离子。无机工业用作生产其他镍盐如硫酸镍铵、氧化镍、碳酸镍等的主要原料。另外，仍可用于生产镍镉电池等。 包装和贮存 存于阴凉、通风的库房。远离火种、热源。应和氧化剂分开存放，切忌混储。 氯化镍 化学式及产品介绍 化学式为NiCl2别名：氯化亚镍 氯化镍的性状为绿色结晶性粉末。在潮湿空气中易潮解，受热脱水，在真空中升华，能很快吸收氨。溶于乙醇、水和氢氧化铵，其水溶液呈酸性，pH约4。 二、作用和用途 氯化镍主要用作电镀和催化剂，由镍和硫硝混酸反应得到。 三、包装和贮存 密封阴凉干燥保存。 氨基磺酸镍 化学式及产品介绍 化学式为Ni（NH2SO3)2.4H2O 氨基磺酸镍的性状呈绿色结晶，易溶于水、液氨、乙醇，微溶于丙酮。水溶液呈酸性,有吸湿性,潮湿空气中很快潮解。干燥空气中缓慢风化,受热时会失去四个分子水,温度高于110时开始分解且形成碱式盐,继续加热生成棕黑色的三氧化二镍和绿色的氧化亚镍的混合物。 二、作用和用途 氨基磺酸镍是壹种优良的电镀主盐,因其内应力低、电镀速度快、溶解度大、无污染等,而成为近年国际上发展较快的壹种电镀主盐。已广泛应用于冶金、镍网、电子、汽车、航天、兵器、造币、无线电、彩色铝合金等行业。 三、包装和贮存 贮存于通风、干燥的库房中。包装必须完整密封,注意防潮。运输过程中要防雨淋和日光曝晒。消泡剂 分子式及产品介绍 破泡剂·抑泡剂·脱泡剂总称为消泡剂。在工业生产的过程中会产生许多有害泡沫，需要添加消泡剂。消泡剂的种类很多，有机硅氧烷、聚醚、硅和醚接枝、含胺、亚胺和酰胺类的，具有消泡速度快，抑泡能力强的特性。 二、作用和用途 消泡剂广泛应用于线路板、工业清洗、清除胶乳、纺织上浆、食品发酵、生物医药、涂料、石油化工、造纸等行业生产过程中产生的有害泡沫。 三、包装和贮存 密封，放置在阴凉干燥处远离火源。 氢氧化钠 化学式及产品介绍

超材料(metamaterials)在电子元件中的应用

第 27 卷 第 9 期 2008 年 9 月
电 子 元 件 与 材 料 ELECTRONIC COMPONENTS AND MATERIALS
Vol.27 No.9 Sep. 2008
新一代片式元件
超材料(metamaterials)在电子元件中的应用
周 济
（清华大学 材料科学与工程系, 北京 100084） 摘要: 超材料（metamaterials）指的是一些呈现出天然材料所不具备的超常物理性质的人工复合材料。它们的超 常特征来源于其中人工制备的、特殊的非均匀插入结构所导致的物理响应。介绍了近年来发展出的超材料包括左手材 料、 “隐身斗篷”和光子晶体等，对其在电子元件领域中的应用进行了评述和展望。 关键词: 电子技术；超材料；综述；电子元件；左手材料 中图分类号: TB39 文献标识码:A 文章编号:1001-2028（2008）09-0001-04
Applications of metamaterials in electronic components
ZHOU Ji
(Department of Materials Science and Engineering, Tsinghua University, Beijing 100084,China) Abstract: A new class of artificial composites that exhibit exceptional properties not readily observed in nature called metamaterials. These properties arise from qualitatively new response functions that are not observed in the constituent materials and result from the inclusion of artificially fabricated, extrinsic inhomogeneities. A few metamaterials being developed in recent years, including left-handed materials, invisible cloak and photonic crystals were summarized. Review and prospect on applications of metamaterials in the area of electronic components were presented. Key words: electron technology; metamaterials; review; electronic components; left-handed materials
metamaterial（超材料）是 21 世纪物理学领域出 现的一个新的学术词汇，近年来经常出现在各类科学 文献中。拉丁语“meta-”，可以表达“超出、亚、另类” 等含义。对于 metamaterial 一词，目前尚未有一个严 格的、权威的定义，不同的文献上给出的定义也各不 相同，但一般都认为 metamaterial 是“具有天然材料所 不具备的超常物理性质的人工复合结构或复合材料”。 在互联网上颇有影响的维基百科（Wikipedia）上，对 metamaterial 一词是这样解释的：“In electromagnetism (covering areas like optics and photonics), a meta material (or metamaterial) is an object that gains its (electromagnetic) material properties from its structure rather than inheriting them directly from the materials it is composed of. This term is particularly used when the resulting material has properties not found in naturally-formed substances”。这一解释可能是迄今对 metamaterial 一词给出的最科学规范的定义， 尽管这一 定义从目前的观点看过于狭隘（该定义似乎只针对电 磁领域的材料，而实际上，最新的研究表明 metamaterial 已经包括一些声学材料） 。 从这一定义中， 我们可以看到 metamaterial 的三个重要特征：
（1）metamaterial 通常是具有新奇人工设计结构 的复合材料； （2）metamaterial 具有超常的物理性质（往往是 自然界的材料所不具备的） ； （3）metamaterial 的性质往往不主要取决于构成 材料的本征性质， 而主要取决于其中的人工设计结构。 目前，人们已经发展出的这类超材料系统包括光 子晶体、左手材料、“隐身斗篷（invisible cloak）”和 全吸收超材料等。 近年来，各种超材料系统的出现引起了科学界的 广泛关注。1998 年和 1999 年，与光子晶体有关的研 究新突破先后两度被 Science 杂志列为世界上的“十大 科学进展”，2006 年底，该杂志又再次将光子晶体列 为未来自然科学的热点领域。而另一类超材料——左 手材料则是在 2003 年被 Science 杂志列为当年的“ 十 大科学进展”。2006 年底，由于英美两国科学家利用 与左手材料相类似的设计方法获得的梯度超材料成功 实现了“隐身斗篷”的功能， Science 杂志又一次将其列 为当年的“ 十大科学进展”。笔者将着重对近年来超材 料在电子元件领域的应用及发展动向做一简要介绍。
收稿日期：2008-07-31 作者简介 ： 周济 （1962－） ， 男， 吉林九台人， 长江特聘教授， 博士后， 主要从事信息功能材料研究。 Tel:(010) 62772975； E-mail: zhouji@https://www.wendangku.net/doc/c01120385.html, 。

半导体材料

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Instrumentation and Equipments 仪器与设备, 2019, 7(2), 133-141 Published Online June 2019 in Hans. https://www.wendangku.net/doc/c01120385.html,/journal/iae https://https://www.wendangku.net/doc/c01120385.html,/10.12677/iae.2019.72019 Research Progress of Metamaterial Absorber Jiali Chai, Yanjie Ju* School of Electrical and Information Engineering, Dalian Jiaotong University, Dalian Liaoning Received: Jun. 3rd, 2019; accepted: Jun. 21st, 2019; published: Jun. 28th, 2019 Abstract In order to make better use of electromagnetic waves and eliminate their negative effects, meta-material absorbers have become a major research direction. This is a device that converts elec-tromagnetic wave energy incident on its surface into other energy to deplete it through special structures and materials. Its particularity based on the application of metamaterials, and its unique electromagnetic properties compared with natural materials make it has great signific-ance in the electromagnetic field. In this paper, the current research status of supermaterial ab-sorbers at home and abroad will be introduced through the structures, mechanisms and materials of the absorbers. For the structures, it mainly introduces two types of tiled-array structure and three-dimensional structure. For the absorption mechanisms, it mainly introduces the frequency selection surface, electromagnetic resonance and surface plasma. For the materials, it introduces metal materials, ferrite materials, carbon materials and new materials in detail. With the conti-nuous innovation in the field of materials and the unremitting efforts of researchers, we believed the absorbing device will be applied to more and more fields with more perfect performances and shine in both the civilian and military fields. Keywords Metamaterials, Absorber, Graphene, Absorbing Mechanism 超材料吸波器的研究进展 柴佳丽，鞠艳杰* 大连交通大学电气信息工程学院，辽宁大连 收稿日期：2019年6月3日；录用日期：2019年6月21日；发布日期：2019年6月28日 *通讯作者。

半导体材料有哪些

半导体材料有哪些 半导体材料有哪些 半导体材料很多，按化学成分可分为元素半导体和化合物半导体两大类。锗和硅是最常用的元素半导体;化合物半导体包括第Ⅲ和第Ⅴ族化合物（砷化镓、磷化镓等）、第Ⅱ和第Ⅵ族化合物（硫化镉、硫化锌等）、氧化物（锰、铬、铁、铜的氧化物），以及由Ⅲ-Ⅴ族化合物和Ⅱ-Ⅵ族化合物组成的固溶体（镓铝砷、镓砷磷等）。除上述晶态半导体外，还有非晶态的玻璃半导体、有机半导体等。 半导体的分类，按照其制造技术可以分为：集成电路器件，分立器件、光电半导体、逻辑IC、模拟IC、储存器等大类，一般来说这些还会被分成小类。此外还有以应用领域、设计方法等进行分类，虽然不常用，但还是按照IC、LSI、VLSI（超大LSI）及其规模进行分类的方法。此外，还有按照其所处理的信号，可以分成模拟、数字、模拟数字混成及功能进行分类的方法。 延伸 半导体材料是什么？ 半导体材料（semiconductor material）是一类具有半导体性能（导电能力介于导体与绝缘体之间，电阻率约在1mΩ·cm～1GΩ·cm范围内）、可用来制作半导体器件和集成电路的电子材料。 自然界的物质、材料按导电能力大小可分为导体、半导体和绝缘体三大类。半导体的电阻率在1mΩ·cm～1GΩ·cm范围（上限按谢嘉奎《电子线路》取值，还有取其1/10或10倍的；因角标不可用，暂用当前描述）。在一般情况下，半导体电导率随温度的升高而升高，这与金属导体恰好相反。 凡具有上述两种特征的材料都可归入半导体材料的范围。反映半导体半导体材料内在基本性质的却是各种外界因素如光、热、磁、电等作用于半导体而引起的物理效应和现象，这些可统称为半导体材料的半导体性质。构成固态电子器件的基体材料绝大多数是半导体，正是这些半导体材料的各种半导体性质赋予各种不同类型半导体器件以不同的功能和特性。 半导体的基本化学特征在于原子间存在饱和的共价键。作为共价键特征的典型是在晶格结构上表现为四面体结构，所以典型的半导体材料具有金刚石或闪锌矿（ZnS）的结构。由于地球的矿藏多半是化合物，所以最早得到利用的半导体材料都是化合物，例如方铅矿

新型半导体材料GaN简介

新型半导体材料GaN GaN的发展背景 GaN材料的研究与应用是目前全球半导体研究的前沿和热点，是研制微电子器件、光电子器件的新型半导体材料，并与SIC、金刚石等半导体材料一起，被誉为是继第一代Ge、Si半导体材料、第二代GaAs、InP化合物半导体材料之后的第三代半导体材料。它具有宽的直接带隙、强的原子键、高的热导率、化学稳定性好（几乎不被任何酸腐蚀）等性质和强的抗辐照能力，在光电子、高温大功率器件和高频微波器件应用方面有着广阔的前景。 在宽禁带半导体材料中，氮化镓由于受到缺乏合适的单晶衬底材料、位错密度大等问题的困扰，发展较为缓慢，但进入90年代后，随着材料生长和器件工艺水平的不断发展，GaN半导体及器件的发展十分迅速，目前已经成为宽禁带半导体材料中耀眼的新星。 GaN的特性 具有宽的直接带隙、强的原子键、高的热导率、化学稳定性好（几乎不被任何酸腐蚀）等性质和强的抗辐照能力，在光电子、高温大功率器件和高频微波器件应用方面有着广阔的前景。 GaN是极稳定的化合物，又是坚硬的高熔点材料，熔点约为1700℃，GaN 具有高的电离度，在Ⅲ—Ⅴ族化合物中是最高的（0.5或0.43）。在大气压力下，GaN晶体一般是六方纤锌矿结构。它在一个元胞中有4个原子，原子体积大约为GaAs的一半。因为其硬度高，又是一种良好的涂层保护材料。在室温下，GaN 不溶于水、酸和碱，而在热的碱溶液中以非常缓慢的速度溶解。NaOH、H2SO4和H3PO4能较快地腐蚀质量差的GaN，可用于这些质量不高的GaN晶体的缺陷检测。GaN在HCL或H2气下，在高温下呈现不稳定特性，而在N2气下最为稳定。GaN的电学特性是影响器件的主要因素。未有意掺杂的GaN在各种情况下都呈n 型,最好的样品的电子浓度约为4×1016/cm3。一般情况下所制备的P型样品，都是高补偿的。 很多研究小组都从事过这方面的研究工作，其中中村报道了GaN最高迁移率数据在室温和液氮温度下分别为μn=600cm2/v·s和μn=1500cm2/v·s，相应的载流子浓度为n=4×1016/cm3和n=8×1015/cm3。未掺杂载流子浓度可控制在

化工材料知识

名称：丁胺黑药 主要成份：二丁基二硫代磷酸铵 分子式：（C4H9O）2PSSNH4 性状：白色至灰白色粉末，无味，在空气中潮解，溶于水，化学性质稳定。 主要用途：丁胺黑药是有色金属硫化矿的优良捕收剂，有一定兼起泡性。对铜、铅、银及活化了的锌硫化矿以及难选多金属矿有特殊的分选效果，它在弱碱性矿浆中对黄铁矿和磁黄铁矿的捕收性能较弱，而对方铅矿的捕收能力较强。它也可用于镍、锑硫化矿的浮选，特别对难选的硫化镍矿、硫化一氧化镍混合矿以及硫化矿与脉石的中矿较为有效。根据研究，使用丁胺黑药还有利于提高铂、金、银的回收。 聚合氯化铝 聚合氯化铝是一种无机高分子混凝剂，又被简称为聚铝，英文缩写为PAC，用于氢氧根离子的架桥作用和多价阴离子的聚合作用而生产的分子量较大、电荷较高的无机高分子水处理药剂。在形态上又可以分为固体和液体两种，而固体按颜色不同又分为棕褐色、黄色和白色，不同颜色的聚合氯化铝在应用及生产技术上也有较大的区别。 聚丙烯酰胺简称PAM，又分阴离子（HPAM)阳离子(CPAM)，非离子(NPAM)是一种线型高分子聚合物，是水溶性高分子化合物中应用最为广泛的品种之一，聚丙烯酰胺和它的衍生物可以用作有效的絮凝剂、增稠剂、纸张增强剂以及液体的减阻剂等，广泛应用于水处理、造纸、石油、煤炭、矿冶、地质、轻纺、建筑等工业部门。性能特点： 1、聚丙烯酰胺分子中具有阳性基因，絮凝能力强，用量少，处理效果明显。 2、溶解性好，活性高，在水体中凝聚形成的矾花大，沉降快，比其他水溶性高分子聚合物净化能力大2-3倍。 3、适应性强受水体PH值和温度影响小，原水净化后达到国家引用水标准，处理后水中悬浮颗粒达到絮凝澄清的目的，有利于离子交换处理和高纯水的制备。 4、腐蚀性小，操作简便，能改善投药工序的劳动强度和劳动条件。 漂白粉是由氯气与氢氧化钙（消石灰）反应而制得。中文名称：次氯酸钙。用于漂白或消毒作用。

单负超材料简介

单负材料具有一些特殊性质，因而受到广泛关注。他的双层结构可以有效成双负材料，且这种双层结构有许多有趣的性质：共振，透明，反常的隧道效应和零反射率。由单负材料构成的一维光子晶体能够形成一种具有较强稳定性的光子带隙，因而能够突破传统的衍射极限，实现次波长成像。含此类单负材料的多层结构体系不仅能够实现远距离成像，而且能够较大幅度地提高体系成像质量。 2010年，美国研究人员又由由超材料纳米线阵列开发出了一种新型纳米镜头，打破了衍射极限，获得了现有技术尚无法达到的所谓超高分辨率成像。此项研究成果发表在了2010 年的《应用物理快报》（ AppliedPhysics Letters ）上。 2011年，随着信息技术的快速发展,现代高新技术也都在向着更加精细的领域发展"尤其是对于高端纳米光学成像技术应用,如光学光刻!共聚焦显微技术!高密度光存储!纳米激光加工!生物显微成像以及生命科学等领域,常常需要有亚波长(纳米量级)的分辨本领" 然而,由于衍射极限的存在,传统光学成像技术己经不能满足实际的要求"本文基于突破传统衍射光学极限的亚波长超分辨率成像技术一双曲透镜技术,通过将传统的提高光刻分辨率技术一相移掩膜技术(phaseshiftmask,PSM)与超级透镜技术相结合,提出了一种超分辨率纳米光刻成像系统"理论分析和数值仿真表明此系统能够大幅度提高现有光刻技术的分辨率"同时,基于一种具有天然材料所不具备的超常物理性质,且其特性可根据需要人为调节的超常材料,设计了一种可实现亚波长聚焦的喇叭聚光镜"这种能够工作在不同工作波长下,聚焦光斑可以达到几个纳米的超透镜将有着重要的潜在应用价值。 另一方面，声波超材料是一种人造复合材料,通过设计组分单元的谐振,在波动载荷(声波)作用下,其在宏观等效意义匕具有传统材料所不具备〔或很难具备)的物理属性:如负等效质量、各向异性等效质量以及负等效模量等。大多数声波超材料都是负折射率超材料。声波在该类材料中传播会表现出奇异的频散特性。研究发现.经过特殊设计的声波超材料可以突破衍射极限的限制,实现声波高分辨率成像.在生物医学成像、上业无损检测等领域具有厂阔的应用前景。 基于质量弹簧模型,负等效质量的形成机制和频散特征【l],深入研究发现了服从Drude模型的声波超材料,其等效质量在某一截止频率以下均为负值【21】。在此基础上,设计出了由金属网格填充软橡胶组成的超材料,实腾测试证明在负质量频带具有良好的低频隔声性能[3]。接着，研究零质量现象，发现在其对应频率声波具有全透射功能,并且对凋落波也具有传输作用。基于该特性，设计出了具有各向异性等效质量的平板声透镜【4〕,其中平行和垂直界面方向的等效质量分别为无穷大和零,无穷大质量用于将凋落波转化为行进波,数值模拟结果表明所设计超材料透镜可以分辨物体的亚波长信息。进一步研究发现,在该模型中还存在共振遂穿效应,在遂穿频率也具有超分辨成像功能〔5〕。实验上,设计并制备出了基于共振遂穿效应的多孔平板透镜,与基于法布里一波罗共振机理的透镜相比,成像频率可以通过内部孔洞的孔径比调节.而与透镜厚度无关,实验结果表明所制备透镜可以分辨间趾小于衍射极限的两个声源。 近几年来，对于超材料在隐身领域的研究也受到了广泛的关注［3- 5 ］。由于超材料可实现与以前常规材料截然不同的折射，因此人们对隐身的研究注意力也从单纯的吸波研究扩展到了控制电磁波的绕射从而达到隐身的目的。基于