3RNA-directed DNA methylation
Marjori Matzke,Tatsuo Kanno,Bruno Huettel,Estelle Jaligot, M.Florian Mette,David P.Kreil,Lucia Daxinger,Philipp Rovina,Werner Aufsatz and Antonius J.M.Matzke
3.1Introduction
3.1.1RNA interference
RNA interference(RNAi)has provided a new paradigm for understanding gene regulation in many eukaryotic organisms.Although plant scientists laid much of the groundwork for the discovery of RNAi(Matzke&Matzke,2004), it was the identification of double-stranded RNA as the trigger for gene silencing in Caenorhabditis elegans by Fire et al.(1998)that provided the means to downregulate gene expression reproducibly in plants,animals and many fungi.The canonical RNAi pathway is now well established:double-stranded RNA is processed by a ribonuclease III–type enzyme called Dicer into short interfering RNAs(siRNAs)of around21–24nt in length.The siRNAs associate with the RNA-induced silencing complex(RISC)and guide cleavage of complementary mRNAs(Novina&Sharp,2004).A second type of small RNA,the microRNA(miRNA),is also produced via Dicer cleavage of longer duplex RNAs.The miRNAs associate with an RISC-like complex and,depending on the degree of complementarity to the target mRNA,elicit either mRNA cleavage or translational repression.In addition to Dicer,other core proteins of the RNAi machinery include Argonaute,an RISC component that binds small RNAs and in some cases executes mRNA cleavage,and RNA-dependent RNA polymerase(RDR),which can synthesize double-stranded RNA from single-stranded RNA templates to initiate or amplify the RNAi reaction(Bartel,2004;He&Hannon,2004).
In addition to making substantial contributions to elucidating the classical RNAi pathway,plant scientists have been at the forefront of research on RNA-mediated epigenetic modifications.Epigenetics refers to mitotically and/or meiotically heritable changes in gene expression that do not involve a change in DNA sequence.Epigenetic modifications include DNA cytosine(C)methy-lation and various posttranslational modifications of histones,including acet-ylation and methylation.The connection between RNAi and epigenetic regulation is exemplified by the phenomenon of RNA-directed DNA methy-lation(RdDM),which is the subject of this chapter.
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3.1.2Discovery and characteristics of RNA-directed DNA methylation Originally detected in viroid-infected tobacco plants,RdDM was the first RNA-guided epigenetic modification of the genome to be reported(Wasse-negger et al.,1994).Viroids are minute plant pathogens consisting exclusively of a non-protein-coding,circular rod-shaped RNA several hundred base pairs in length(Tabler&Tsagris,2004).In the experiments that revealed RdDM,viroid cDNAs integrated as transgenes into tobacco chromosomes became methylated de novo during RNA–RNA replication of the cognate viroid.Further analysis demonstrated that Cs in all sequence contexts are modified and that methylation is largely confined to the region of RNA–DNA sequence homology(Pe′lissier et al.,1999).DNA regions as short as30bp can be targeted for methylation in the RdDM pathway(Pe′lissier& Wassenegger,2000).
The identification of RdDM occurred in the framework of homology-dependent gene-silencing phenomena,which were postulated at the time to be due variously to DNA–DNA,DNA–RNA or RNA–RNA interactions (Jorgensen,1992;Matzke&Matzke,1993).Indeed,one of the initial papers describing‘cosuppression’,a process in which a resident gene is silenced in the presence of a homologous transgene,proposed that transgene RNA might interact with DNA of the resident gene to block transcription(van der Krol et al.,1990).Even though cosuppression turned out to be a posttran-scriptional gene-silencing phenomenon that is now considered the plant equivalent of RNAi(De Carvalho et al.,1992;Matzke&Matzke,2004; van Blokland et al.,1994),this example illustrates that RNA–DNA inter-actions were considered as a possible trigger of silencing during the early stages of gene-silencing research.The viroid experiments supplied experi-mental documentation of this possibility and revealed the characteristic features of RdDM.Shortly thereafter,cosuppression(also known as post-transcriptional gene silencing(PTGS))of a reporter transgene in tobacco was shown to be accompanied by de novo methylation of the corresponding genomic DNA sequences(Ingelbrecht et al.,1994).Thus,a transgene subject to posttranscriptional regulation could also be targeted for DNA methylation, suggesting that cytoplasmic and nuclear events are induced by a common silencing mechanism.
A further connection between PTGS and DNA methylation was observed in transgenic pea plants infected with an RNA virus that replicates exclusively in the cytoplasm.Virus replication initiated PTGS of a transgene encoding the viral replicase gene,which was accompanied by de novo methylation of replicase transgene sequences integrated into nuclear DNA(Jones et al., 1998).The pattern of methylation was consistent with RdDM,occurring at Cs in both symmetrical(CG and CNG,where N is A,T or C)and asymmet-rical(CNN)sequence contexts within the region of RNA–DNA sequence
RNA-DIRECTED DNA METHYLATION71 homology.These findings suggested that a sequence-specific signal,most likely RNA,produced during virus replication and/or PTGS diffused from the cytoplasm into the nucleus to induce methylation of homologous DNA sequences.In an extension of these experiments,replicating potato virus X RNA vectors modified to contain sequences homologous to the35S promoter were able to trigger methylation and transcriptional silencing of35S pro-moter–driven nuclear transgenes(Jones et al.,1999).
The experiments with viroids and viruses hinted that RdDM is initiated by a double-stranded RNA because these RNA pathogens replicate via a double-stranded RNA intermediate.An unequivocal demonstration that double-stranded RNA is required for RdDM came from studies on a non-pathogenic,transgenic system.These experiments grew from work that suggested a role for RNA in mediating methylation of transgene promoters (Mette et al.,1999;Park et al.,1996).To further investigate this possibility, RNAs that contained promoter sequences were tested for their ability to induce methylation and transcriptional silencing of unlinked homologous promoters in transgenic tobacco plants.Cre-lox-mediated recombination was used to convert a transcribed direct repeat of target promoter sequences into a tran-scribed inverted repeat in planta.Methylation and silencing of a homologous target promoter was observed only after generation of the inverted repeat and initiation of double-stranded RNA synthesis(Mette et al.,2000).The double-stranded RNA that induced RdDM was shown to be processed to short RNAs 21–24nt in length,similar to those involved in RNAi,reinforcing a link between the two silencing processes.Double-stranded RNAs containing promoter sequences have subsequently been used to silence and methylate different transgene promoters in Arabidopsis thaliana(Aufsatz et al.,2002a, 2002b;Kanno et al.,2004)and several endogenous promoters in petunia (Sijen et al.,2001)and Arabidopsis(Melquist&Bender,2003).Therefore, RNA-mediated transcriptional gene silencing(TGS)and promoter methyla-tion appears to be a general process in plants.
3.2RNAi-mediated pathways in the nucleus
Viewed as a plant-specific phenomenon for several years after its discovery, RdDM is now regarded as one of several RNAi-mediated pathways in the nucleus.In addition to RdDM,the other nuclear pathways include:
(1)RNAi-mediated heterochromatin formation that has been observed in
fission yeast,plants,Drosophila melanogaster and vertebrates;
(2)elimination of intergenic DNA that has been packaged into heterochro-
matin via the RNAi pathway during nuclear differentiation in Tetrahy-mena thermophila and other ciliated protozoa;
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(3)meiotic silencing of DNA that is unpaired during meiosis in Neurospora
crassa and C.elegans(reviewed by Matzke&Birchler,2005).
Here we focus on RdDM and on RNAi-mediated heterochromatin formation, particularly as it relates to DNA methylation.
3.2.1RNAi-mediated heterochromatin formation
The involvement of the RNAi pathway in heterochromatin formation was first discovered in fission yeast(Schizosaccharomyces pombe).Mutants defective in the core RNAi proteins–Dicer,Argonaute and RDR–were shown to be unable to assemble functional heterochromatin at the centromeres(Volpe et al., 2002,2003).In wild-type cells,transient double-stranded RNA molecules originating from forward and reverse transcription of centromere outer repeats are processed by Dicer to siRNAs(Reinhart&Bartel,2002).The siRNAs are thought to guide histone H3lysine9(H3K9)methylation that is catalyzed by cryptic loci regulator4(Clr4),the S.pombe ortholog of the histone methyl-transferase Su(var)3–9(from Drosophila suppressor of variegation3–9).Swi6, the S.pombe ortholog of Drosophila heterochromatin protein1(HP1)can bind via its chromodomain to histone H3methylated on K9,which promotes lateral spreading of heterochromatin from the siRNA-targeted nucleation site.The formation of heterochromatin leads to binding of cohesin,which fosters sister chromatid cohesion and proper chromosome segregation.Chromatin-associ-ated RDR primes RNA synthesis from the reverse transcript of the outer centromere repeat,which is continuously transcribed,ensuring a constant supply of double-stranded RNA even when transcription of the forward strand is repressed by heterochromatin formation(reviewed by Maison&Almouzni, 2004).
A similar process occurs at the S.pombe silent mating type locus,which contains a copy of the centromeric outer repeat(Hall et al.,2002).In addition, synthetic hairpin RNAs can induce heterochromatin formation at the sites of genes that are normally euchromatic(Schramke&Allshire,2003).Another natural target of RNAi-mediated heterochromatin in S.pombe is retrotranspo-son long terminal repeats(LTRs)that are transcribed bidirectionally.Spread-ing of heterochromatin from LTRs into adjacent genes can contribute to cellular differentiation by silencing stage-specific gene expression(Schramke &Allshire,2003).A nuclear complex called RNA-induced initiation of transcriptional gene silencing(RITS)has been isolated,which directly links siRNAs to heterochromatin in S.pombe.The RITS complex contains siRNAs from known heterochromatic regions,such as centromeres,as well as Chp1,a chromodomain protein that binds centromeres,the S.pombe ortholog of Ago1, and Tas3,a serine-rich protein that is found only in S.pombe(Verdel et al., 2004).The RITS complex is tethered to silenced loci that contain H3K9
RNA-DIRECTED DNA METHYLATION73 methylation.This tethering faciliates siRNA production and maintenance of heterochromatin(Noma et al.,2004).
The RNAi-mediated heterochromatin pathway is conserved in animals. Heterochromatin in Drosophila,assessed by H3K9methylation and localiza-
tion of the heterochromatin proteins HP1and HP2,is disrupted in mutants defective in piwi and aubergine,which are both members of the PAZ/piwi domain family that contains Argonaute,and in spindle-E(homeless),a DEAD-
box RNA helicase important for RNAi(Pal-Bhadra et al.,2004).In vertebrate cells,formation of centromeric heterochromatin depends on functional Dicer activity.This was shown by generating a conditional loss-of-function Dicer mutant in chicken–human hybrid cells that contain human chromosome21. Aberrant accumulation of transcripts from human centromeric a-satellite repeats and delocalization of two heterochromatin proteins,Rad21cohesin
and the mitotic checkpoint protein BubR1,were observed in the Dicer-deficient cells,which also displayed mitotic defects and frequently died in interphase(Fukagawa et al.,2004).
The most comprehensive study on RNAi-mediated heterochromatin in plants has been carried out on the heterochromatic knob on chromosome4
of A.thaliana.This work is considered in the context of RdDM(see Section
3.2.2AQ1
).
3.2.2RdDM and RNAi-mediated heterochromatin assembly:one pathway
or two?
While RdDM and RNAi-mediated heterochromatin are likely to be inter-related,it is not yet clear whether they are the outcomes of a single pathway
or two separate pathways.Both RdDM and RNAi-mediated heterochromatin formation are initiated by double-stranded RNAs that are substrates of Dicer cleavage,but they might diverge after this step to yield distinct primary epigenetic marks that differ in terms of chemistry,mitotic heritability and reversibility.
Historically,RdDM has been studied from the angle of C methylation, which we assume is the primary epigenetic mark associated with this process.
This assumption is based on the pattern of methylation,which is largely confined to the region of RNA–DNA sequence homology and which can encompass sequences as short as30bp.This contrasts to histone modifications
that occur in a nucleosomal context that comprises147bp of DNA.Often referred to as the‘fifth base’in eukaryotic DNA(Hergersberg,1991),
5-methylcytosine represents a covalent modification of the DNA molecule itself.By contrast,the primary epigenetic mark that is evaluated for RNAi-mediated heterochromatin formation involves covalent modification of histone proteins,most typically methylation of lysine9of histone H3,which is a hallmark of silent heterochromatin.
C methylation is carried out by enzymes known as DNA methyltransferases.The cytosine-5-methyltransferases transfer a methyl group from S-adenosyl-l -methionine to carbon 5of C residues (Finnegan &Kovac,2000).When present in symmetrical CG and CNG nucleotide groups,methylation can be copied faithfully during DNA replication and passed on to daughter cells (see Figure 3.1).In addition to passive loss of methylation,which results from deficiencies in maintenance methylation,there is increasing evidence that CG methylation can be actively removed from non-replicating DNA through the activity of DNA glycosylases in plants (Choi et al .,2002;Gong et al .,2002;Kinoshita et al .,2004)and in vertebrates (reviewed by Kress et al .,2001).Thus,CG and CNG methylation are mitotically heritable but also potentially revers-ible epigenetic modifications.It is not yet clear how histone modifications can be inherited through rounds of DNA replication and cell divisions.Indeed,if the term ‘epigenetic’implies mitotic heritability,then some authors have questioned whether histone modifications fully qualify owing to uncertainties
m m m Dnmt2 (?): vertebrates, plants,
D. melanogaster , S. pombe
RNA
vs maintenance methylation
RNA
DRM1, DRM2/Dnmt3a, b
(MET1; CMT3?)de novo methylation AQ2
Figure 3.1DNA methyltransferases catalyzing de novo and maintenance methylation.Plant enzymes are shown in bold.De novo methylation refers to methylation of a previously unmodified DNA sequence.One of the best-documented signals for de novo methylation is double-stranded RNA that can be processed by Dicer-like activity into short RNAs (dashed lines;not drawn to scale).DNA methyltransferases that are generally classified as de novo activities include mammalian Dnmt3a/b and the plant homologs,DRM1,DRM2.Methylation can be maintained in symmetrical CG (and in plants,CNG)nucleotide groups through subse-quent rounds of DNA replication (arrow)by the action of so-called maintenance methyltransferases,which recognize the methylated C in the parental strand and catalyze methylation of the opposite C in the newly synthesized strand.The CG maintenance activity in mammals is Dnmt1;the plant homolog is MET1.Plants have a special DNA methyltransferase,CMT3,that can maintain methylation in CNG trinucleotides.Recent work on RdDM indicates that MET1and CMT3can also participate in de novo methylation in the presence of RNA signals.Methylation in asymmetric CNNs cannot be maintained and requires the continuous presence of the triggering RNA.Members of the enigmatic Dnmt2class of DNA methyltransferases,whose function is not known,are present in vertebrates and plants and in organisms not normally thought to methylate their DNA,such as Drosophila melanogaster and fission yeast (Schizosaccharomyces pombe ).
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about their inheritance during mitosis (Bird,2002).Moreover,even though arginine methylation in histones can be altered by enzymes that convert methylated arginines to citrullines (Cuthbert et al .,2004;Wang et al .,2004),enzymes that can remove lysine methylation in histones have not yet been identified (Zhang,2004).Thus,despite an incomplete understanding of its mode of inheritance,H3K9methylation is thought to be a means for ensuring relatively stable,long-term silencing.
RdDM and RNAi-mediated heterochromatin might be further distinguished by the manner in which short RNAs interact with the homologous target sequence (see Figure 3.2).The failure of RdDM to spread substantially beyond the region of RNA–DNA sequence homology hints that direct RNA–DNA base pairing provides a substrate for methylation.By contrast,models in which short RNAs base-pair to nascent RNAs transcribed from the target locus have been proposed for RNAi-mediated heterochromatin assembly (Grewal &Moazed,2003),which might somehow facilitate spreading beyond the siRNA-targeted nucleation site.
DNA methylation can influence histone modifications and vice versa,but the order of events appears to vary depending on the system under investiga-tion (Lund &van Lohuizen,2004;Mutskov &Felsenfeld,2004;Tariq &Paszkowski,2004).For example,in N.crassa ,all DNA methylation is cata-lyzed by one DNA methyltransferase,DIM-2(Kouzminova &Selker,2001);this DNA methylation,however,is fully dependent on DIM-5,a histone H3K9methyltransferase (Tamaru &Selker,2001).In Neurospora ,therefore,histone methylation clearly precedes –and is a prerequisite for –DNA methylation.However,Neurospora might be exceptional.The structure of the DIM-2DNA methyltransferase differs from the four major families of DNA
short RNA short RNA
nascent RNA
dsDNA dsDNA RNA polymerase
RNA ? RNA
RNA ? DNA
Figure 3.2Possible modes of interaction between short RNAs and the homologous target locus.Short RNAs can base-pair to a nascent RNA transcribed from the target locus (RNA–RNA)or to one strand of DNA (RNA–DNA).
RNA-DIRECTED DNA METHYLATION 75
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3.3.2.2)(Groll&Bestor,2005).Moreover, methyltransferase(see Section AQ3 unlike many other eukaryotes,Neurospora does not seem to use the RNAi machinery to guide epigenetic modifications(Chicas et al.,2004;Freitag et al., 2004).Instead,signals for de novo DNA methylation are generated by the unusual process of repeat-induced point mutation(RIP),which produces C:G
to T:A transition mutations in duplicated DNA regions by a poorly understood mechanism.‘RIPed’sequences are preferentially targeted for methylation, apparently because of their A:T richness and high density of TA dinucleotides (Tamaru&Selker,2003).
In another example,silencing of the redundant X chromosome in female mammals appears to involve initially methylation of histone H3at Lys9and
Lys27,followed by DNA methylation(Okamoto et al.,2004).In Arabidopsis, there are conflicting reports about whether DNA methylation precedes (Malagnac et al.,2002;Soppe et al.,2002;Tariq et al.,2003)or follows (Gendrel et al.,2002;Jackson et al.,2002;Johnson et al.,2002)histone H3K9 methylation.
Despite these uncertainties and differences among species,it is clear that RNAi-mediated H3K9methylation can be induced in the absence of detect-
able DNA methylation,as is the case in S.pombe.Thus,the mechanisms of DNA methylation and H3K9methylation are not obligatorily coupled.It is conceivable that RdDM represents a distinct pathway that leads only second-
arily to histone modifications.In this chapter,we discuss RdDM as a pathway
of RNA-guided de novo methylation in which histone modifications are imposed in later steps to maintain and/or reinforce C methylation.
3.3Mechanism of RNA-directed DNA methylation:RNA
and protein requirements
3.3.1Systems used for genetic analyses of RdDM
and transcriptional silencing
Genetic investigations performed in our laboratory on RdDM and RNA-mediated transcriptional silencing have exploited well-defined,two-compon-
ent transgene systems in A.thaliana.In these systems,methylation of a transgene promoter is induced experimentally by a double-stranded RNA
that is encoded by a second,unlinked transgene complex(Matzke et al., 2004).We have carried out genetic screens on two promoter systems.The nopaline synthase promoter(NOSpro)is a moderately active,constitutive plant promoter;the a’promoter(a’pro)is a strong seed-specific promoter. Both these promoters are several hundred base pairs in length and have the same overall GC content($45%)but the NOSpro contains about twice as many CG dinucleotides as the a’pro.Thus,one might anticipate a priori that
RNA-DIRECTED DNA METHYLATION77 NOSpro would be more sensitive to CG methylation than the a’pro.Indeed,as described below,the differences in sequence composition between the two promoters are a likely explanation for the different types of mutations that have been recovered for each system.
Although transgenes provide well-defined and manipulatable systems for analysis,it is important to use the knowledge gained with them to understand RNA-mediated transcriptional regulation of endogenous genes.Parallel gen-etic analyses of several endogenous genes that are silenced(or likely to be silenced)by double-stranded RNA–such as SUPERMAN(SUP),PAI2and FWA–are proving particularly informative with regard to RdDM.Before discussing the results of genetic analyses,we briefly describe these three endogenous gene systems.
SUP encodes a zinc finger transcription factor that is important for flower development(Ito et al.,2003).For reasons that are not understood,SUP becomes highly methylated and silenced in ddm1(decrease in DNA methyla-tion1)and methyltransferase1(met1)mutants that otherwise display genome-wide hypomethylation(Jacobsen et al.,2000).The silent loss-of-function ‘epialleles’of SUP,termed the clark kent(clk)alleles,are hypermethylated at Cs in all sequence contexts throughout the transcribed region and at the tran-scription start site(Jacobsen&Meyerowitz,1997).An approximately350-bp region near the5’end is rich in asymmetric CNNs and CNGs,but contains only one CG dinucleotide(Cao&Jacobsen,2002).To facilitate genetic analyses for the identification of loci important for methylation and silencing of SUP,a stable,non-reverting clk allele,clk-st,was created by introducing an additional SUP locus into clk-3plants(Lindroth et al.,2001).The extra SUP sequences at this locus are arranged as an inverted repeat(Cao&Jacobsen, 2002),which is probably transcribed to produce a double-stranded RNA that stabilizes silencing and methylation of the endogenous SUP gene.
The tryptophan biosynthetic(phosphoribosylanthranilate isomerase,PAI) gene family in the Wassilewskija(WS)strain of Arabidopsis comprises four copies of the PAI gene that are densely methylated at CGs and non-CGs throughout their regions of sequence identity.Due to a naturally occurring duplication(perhaps generated by transposons),two copies–PAI1–PAI4–are arranged as an inverted repeat.PAI2and PAI3are unlinked singlet copies.In contrast,ecotype Columbia(Col)has three singlet,unmethylated PAI genes at the analogous loci(Melquist et al.,1999).In WS,only PAI1and PAI2encode functional enzymes but PAI1is the sole expressed copy because it is tran-scribed from an unrelated upstream promoter that is unmethylated.The PAI1–PAI4inverted repeat induces de novo methylation of unmethylated PAI sequences,apparently by encoding a double-stranded RNA that targets the singlet copies for methylation by RdDM(Melquist&Bender,2003,2004). Genetic screens have been carried out for suppressors of hypermethylation and silencing of the PAI2gene.
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The flowering Wageningen(FWA)gene(M.Koornneef,personal commu-nication)encodes a homeodomain transcription factor that displays imprinted (maternal origin–specific)expression in endosperm.Normally FWA is kept silent during the plant vegetative phase by methylation in two transposon-derived direct repeats in the promoter region(Soppe et al.,2000;Lippman et al.,2004).Loss of methylation from the maternal allele in the central cell of the gametophyte through the activity of the DNA glycosylase protein DEMETER leads to maternal expression only in endosperm(Kinoshita et al. 2004).Short RNAs originating from the direct repeats suggest that transcrip-tional repression of FWA occurs by an RNAi-mediated pathway(Lippman et al.,2004).In contrast to SUP,which becomes hypermethylated in ddm1and met1mutants,FWA becomes hypomethylated in these mutants,producing gain-of-function epialleles that condition a late flowering phenotype(Kaku-tani,1997).
3.3.2Steps in the RdDM pathway
The RdDM pathway can be divided into three steps:
(1)synthesis and processing of double-stranded RNA;
(2)de novo methylation of Cs in all sequence contexts in the presence of RNA
signals;
(3)maintenance of CG and CNG methylation in the absence of RNA signals
(or reinforcement of methylation if RNA signals remain available). Forward and reverse genetic approaches are identifying the molecular machin-ery needed for each step.
3.3.2.1Double-stranded RNA synthesis and processing
The issue of double-stranded RNA synthesis and processing is complicated in plants because of:
(1)different ways to synthesize double-stranded RNA,including through the
activity of RDR,which is encoded by a multi-gene family in Arabidopsis;
(2)the existence of multiple Dicer-like(DCL)enzymes and Argonaute
(AGO)proteins that are differentially distributed into nuclear or cytoplas-mic compartments;
(3)the production of two size classes of short RNAs that appear to be
functionally distinct:a longer class that is$24nt in length has been implicated in DNA and histone methylation and a shorter size class that is$21nt in length is involved in the mRNA degradation step of PTGS.
RNA-DIRECTED DNA METHYLATION79 As discussed below,this distinction has generally held up but it is not yet known whether the24-nt class is exclusively capable of eliciting epigenetic modifications in all systems.
Double-stranded RNA can be synthesized by:
(1)bidirectional transcription of double-stranded DNA,producing sense and
antisense RNAs that can anneal with each other;
(2)transcription through an inverted DNA repeat,which forms an RNA that is
self-complementary and can fold back on itself to form a hairpin RNA;
(3)transcription of a single-stranded RNA template,which requires the ac-
tivity of an RDR(see Figure3.3).
Only the third way of producing double-stranded RNA is compromised by mutations in genes encoding RDRs.The A.thaliana genome contains at least three expressed genes that encode RDRs:RDR1,RDR2and RDR6(Xie et al., 2004).Forward genetic screens have demonstrated that RDR6(SGS2/SDE1) is required for PTGS triggered by sense transgenes(Dalmay et al.,2000; Mourrain et al.,2000).Mutations in RDR6reduce DNA methylation associ-ated with PTGS of sense transgenes but not inverted repeat transgenes(Be′clin et al.,2002;Dalmay et al.,2000;Mourrain et al.,2000).Reverse genetics approaches have revealed that RDR2is required for DNA methylation and H3K9methylation of several endogenous repeats and for accumulation of siRNAs originating from these repeats(Chan et al.,2004;Xie et al.,2004). Mutations of RDR1had no effect on the accumulation of these endogenous siRNAs and its function is unknown.
The Arabidopsis genome encodes four DCL activities(Schauer et al., 2002).Three of these–DCL1,DCL2and DCL3–appear to be localized predominantly in the nucleus as assessed by the subcellular distribution of DCL–GFP fusion proteins(Papp et al.,2003;Xie et al.,2004).Forward genetic screens for mutants defective in RNA-mediated TGS of transgene promoters have not yet recovered mutants defective in any of the four DCL enzymes.This may be due to redundancy of these proteins,as has been observed for the two Dicers in Neurospora(Catalanotto et al.,2004).Another possibility is that unprocessed double-stranded RNA can participate to some extent in RdDM.Indeed,we have not yet found a way to eliminate siRNAs originating from the NOSpro or a’pro,which would allow us to confirm they are needed for RdDM in these systems.Neither RdDM of a target NOSpro nor accumulation of NOSpro siRNAs is impaired in dcl1partial loss of function mutants(Papp et al.,2003).Accumulation of siRNAs generated from hairpin RNAs that induce PTGS is also not reduced in dcl1partial loss of function mutants(Finnegan et al.,2003).Instead,the primary role of DCL1appears to be the processing of miRNA precursors(Park et al.,2002;Reinhart et al., 2002).Whether this is the only function of DCL1is unknown.
Reverse genetics has established a role for DCL3in generating 24-nt small RNAs that are important for DNA and H3K9methylation (Chan et al .,2004;Xie et al .,2004).Whether DCL3is the only DCL activity involved in producing short RNAs that elicit epigenetic modifications is unclear (see Figure 3.3).DCL3lacks double-stranded RNA-binding domains,which are present in the other three DCLs AQ5of Arabidopsis (Schauer et al .,2002).DCL3might act as a heterodimer with another DCL activity or in a complex with another double-stranded RNA-binding protein.Despite its apparent nuclear location,DCL2has been implicated in producing siRNAs from RNA viruses that replicate in the cytoplasm (Xie et al .,2004).No function has yet been
de novo
N
MET1maintenance/
reinforcement
CMT3
KYP/SUVH4
Figure 3.3Pathway of RNA-directed DNA methylation as determined from genetic AQ4
analyses in Arabidop-sis .Left:Double-stranded RNA can be synthesized in several ways including transcription of both DNA strands,producing overlapping sense (S)and antisense (AS)transcripts;transcription of an inverted DNA repeat,producing a hairpin RNA;or through the activity of RNA-dependent RNA polymerase (RDR)on a single-stranded RNA template.Double-stranded RNAs are processed by a dicer-like (DCL)activity to generate short RNAs (dashed lines).RDR2has been implicated together with nuclear-localized (N)DCL3in a nuclear pathway that produces 24-nt short RNAs that elicit DNA and histone methylation.Whether this pathway applies to all cases of RdDM is not yet known.Right:In response to RNA signals,site-specific DNA methyltransferases (MET1for CGs and DRM1,DRM2for CGs and non-CGs)cooperate to catalyze an intermediate level of de novo methylation (half-filled circles).Methylation can be either maintained (àRNA)or reinforced (tRNA)by distinct DNA methyltransferases and histone-modifying enzymes,which team up to preserve specific patterns of cytosine methylation.HDA6and MET1maintain primarily CG methylation;CMT3and KYP/SUVH4maintain primarily CNG https://www.doczj.com/doc/6a11271766.html,N methylation cannot be maintained in the absence of the trigger RNA.Two SNF2-like proteins,DRD1and DDM1,are involved in de novo and maintenance methylation,respectively.AGO1and nuclear localized (N)AGO4are thought to bind to short RNAs and facilitate their interaction with the homologous target locus (DNA or nascent RNA;see Figure 3.2).
80PLANT EPIGENETICS
RNA-DIRECTED DNA METHYLATION81 assigned to DCL4,which contains a predicted nuclear localization signal (Schauer et al.,2002).
Argonaute proteins are core components of RISC(Carmell et al.,2002), RITS(Noma et al.,2004;Verdel et al.,2004),and presumably other silencing effector complexes.As members of the PAZ/piwi domain family,Argonaute proteins are able to bind short RNAs through their PAZ domains(Lingel et al., 2003;Yan et al.,2003a)and they are thought to‘shepherd’short RNAs to their site of action(Carmell et al.,2002).At least one Argonaute protein, human Ago2,contains an RNaseH-like domain that catalyzes cleavage of target mRNAs and hence corresponds to the long-sought‘slicer’activity in the RNAi pathway(Liu et al.,2004;Meister et al.,2004).The Arabidopsis genome encodes ten AGO proteins(Morel et al.,2002).Four of these proteins have been assigned a function and two–AGO1and AGO4–are involved in pathways leading to epigenetic modifications(see Figure3.3).
AGO1,the founding member of the Argonaute family(Bohmert et al.,1998), is required for PTGS and DNA methylation mediated by sense transgenes (Beclin et al.,2002;Boutet et al.,2003)as well as the miRNA pathway and plant development(Vaucheret et al.,2004).AGO1also helps to regulate silencing and epigenetic modifications of a small subset of transposons in Arabidopsis(Lippman et al.,2003).AGO4,a nuclear protein(Xie et al., 2004),is proposed to be specialized for RNA-mediated chromatin modifica-tions.Indeed,AGO4–identified as a suppressor of SUP silencing and hyper-methylation–is the only classical RNAi protein recovered in a forward genetic screen of a presumed RNAi-mediated TGS system(Zilberman et al.,2003). AGO4,together with RDR2and DCL3,is also needed for maintenance of DNA methylation at several endogenous repetitive loci in Arabidopsis(Xie et al., 2004)and for de novo methylation of an FWA transgene(Chan et al.,2004).
A mutation in AGO4does not impair de novo methylation triggered by inverted repeats but does reduce maintenance methylation of these sequences(Zilberman et al.,2004).Two other AGO proteins for which functional information is available have not yet been implicated in epigenetic regulation but are important for plant development:ZIP/AGO7(ZIPPY)controls vegetative phase changes (Hunter et al.,2003)and PNH/ZLL/AGO10(PINHEAD/ZWILLE)is required for meristem maintenance(Lynn et al.,1999;Moussian et al.,1998,2003).
It is not yet known whether both sense and antisense RNAs are needed for https://www.doczj.com/doc/6a11271766.html,parable amounts of each polarity are observed on Northern blots of short RNAs in the NOSpro and a’pro systems(Aufsatz et al.,2002a,2002b; Kanno et al.,2004;Mette et al.,2000).Moreover,similar numbers of sense and antisense NOSpro short RNAs were cloned from preparations of size-fractioned RNAs(Papp et al.,2003).miRNAs accumulate predominantly in the antisense orientation owing to stabilization by base pairing to target mRNA and/or preferred incorporation of antisense miRNAs into silencing effector complexes. The equivalent accumulation of NOSpro short RNAs of both polarities would be
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consistent with stabilization by base pairing to both strands of DNA,although this proposal remains to be substantiated experimentally.Sense and antisense small RNAs of the24-nt size class accumulate from different retroelements in tobacco and Arabidopsis(Hamilton et al.,2002).These include the Arabidopsis SINE element AtSN1,which appears to be a target for small RNA-induced DNA and histone methylation(Hamilton et al.,2002;Lippman et al.,2003;Xie et al., 2004;Zilberman et al.,2003,2004).Considering the possibility that small RNAs elicit epigenetic modifications by base pairing to DNA(see Figure3.2), a single polarity should in principle be sufficient to nucleate methylation in the homologous DNA region provided‘maintenance’methyltransferases are avail-able(see Figure3.4).However,RdDM might be more efficient if small RNAs of both orientations are present.On the other hand,the recent report indicating that miRNAs can trigger DNA methylation downstream of the miRNA complemen-tary region(Bao et al.,2004)might be more consistent with an RNA recognition model,in which the antisense miRNA base pairs to a nascent RNA(see Figure 3.2).This may lead to DNA methylation in the vicinity of the miRNA–RNA duplex,which does not necessarily correspond to the region RNA–DNA se-quence homology.Whether this‘off-target’methylation is the result of unique capabilities of miRNAs as compared with siRNAs in inducing DNA methyla-tion or whether it reflects the spreading of epigenetic modifications associated with the RNA recognition model(see Section3.2.2)is not known.Determining the way(s)in which various kinds of short RNA interact with homologous target loci to elicit epigenetic modifications remains one of the most pressing ques-tions in the field(see Section3.5).
3.3.2.2DNA methyltransferases and histone-modifying enzymes
DNA methyltransferases have been classified traditionally according to whether they catalyze de novo or maintenance methylation.De novo methy-lation refers to methylation of a previously unmodified DNA sequence.Main-tenance methylation involves the perpetuation of methylation in symmetric CG(and in plants,CNG)nucleotide groups during successive rounds of DNA replication(see Figure3.1).In animals,methylation is generally thought to be restricted to CG dinucleotides.In plants,however,the situation is more complex because methylation is frequently observed in Cs in all sequence contexts(Meyer et al.,1994),which is the pattern observed in cases of RdDM. As genetic approaches are used to identify DNA methyltransferases required for RdDM,it is increasingly clear that the strict division of these enzymes into de novo and maintenance activities is untenable.Therefore,in the following discussion,we categorize DNA methyltransferases on the basis of their site specificity;in other words,whether they catalyze CG or non-CG methylation. Because methylation in asymmetric CNNs cannot be maintained in the ab-sence of the RNA trigger(see Figure3.1),it is considered here to be a measure of continuous de novo methylation.
There are four major families of C-5DNA methyltransferases in eukaryotes (Groll &Bestor,2005)and plants contain representatives of them all (Finne-gan &Kovac,2000)(see Figure 3.1):
(1)The MET1family comprises homologs of the mouse Dnmt1methyl-transferase,which is typically described as CG maintenance methyltransferase although it displays de novo activity in vitro (Groll &Bestor,2005).There are at least four members of this gene family in Arabidopsis ,with MET1being the most highly expressed representative.
(2)The domains-rearranged (DRM)family,which contains two members in Arabidopsis ,is related to the mouse Dnmt3group of methyltransferases that are usually ascribed a de novo function (Cao et al .,2000).DRM2is the major
CnCn CG nnCnnn CNG nnnnnn CG nnnnC DRM1, 2
CnCn CG nnCnnn CNG nnnnnn CG nnnnC
gngn GC nngnnn GNC nnnnnn GC nnnng m m m m
m m m
MET1MET1
CMT3MET1MET1
CMT3A B C
24 nt RNA
gngn nngnnn GNC nnnng
AQ6
Figure 3.4Cooperation between DRM1,DRM2and MET1to catalyze de novo methylation.(A)A short RNA (shown here as 24nt in length)base pairs to the complementary (top)DNA strand and DMR1,DMR2catalyze de novo methylation of Cs in all sequence contexts within the region of RNA–DNA sequence homology.(B)The resulting hemimethylated CGs and CNGs are then recognized by MET1and CMT3,respectively,which catalyze methylation of the opposite Cs on the bottom DNA strand.This can occur on non-replicating DNA in the absence of the complementary RNA signal and is essentially de novo methylation of the bottom DNA strand.The DNA is now fully methylated at CGs and CNGs (bold),whereas CNN methylation is still limited to the top strand.(C)During DNA replication (leftward arrow),methylation at CGs and CNGs will be maintained by MET1and CMT3,respectively,while all CNN methylation is lost in the absence of RNA.This hypothetical model provides an explanation for the de novo activity of MET1in the presence of RNA signals,which is related to its conventional maintenance activity that relies on recognition of a hemimethylated substrate during DNA replication.
RNA-DIRECTED DNA METHYLATION 83
84PLANT EPIGENETICS
activity in Arabidopsis and has been reported to catalyze methylation of Cs in all sequence contexts(Cao&Jacobsen,2002).There are noteworthy distinc-tions between mammalian Dnmt3and plant DRM enzymes.First,the arrange-ment of ten motifs in the C-terminal catalytic domain differs.In Dnmt3,the order is I–X;in DRM,the order is VI–X,followed by I–V.Despite the rearrangement,DRM and Dnmt3are predicted to fold into similar structures that are equally efficient in catalyzing C methylation.In addition,the N termini of the DRM class contains several ubiquitin-associated domains,which might be involved in ubiquitin binding and proteosome degradation pathways.In contrast,the N terminus of Dnmt3comprises a PWWP domain(a module of 100–150amino acids containing a conserved proline–tryptophan–tryptophan–proline motif)and cysteine-rich domains(Cao et al.,2000).The significance of these differences between the otherwise highly related Dnmt3and DRM DNA methyltransferases is not known.
(3)The chromomethylases(CMTs)are a plant-specific family of C methyl-transferases that contain a chromodomain(chromatin organization modifier), which is a highly conserved motif found in several chromatin associated proteins.Initially found in Drosophila HP1and Polycomb proteins,the$50 amino acid chromodomain binds to histones methylated at different sites (Brehm et al.,2004).The Arabidopsis genome contains three CMT genes. The major activity,CMT3,catalyzes methylation primarily in CNG trinucleo-tides(Bartee et al.,2001;Lindroth et al.,2001).
(4)The Dnmt2family is the most phylogenetically widespread,conserved and enigmatic group of DNA methyltransferases(Groll&Bestor,2005).No function has been assigned to any Dnmt2protein,including the single one encoded in the Arabidopsis genome.Dnmt2homologs are present even in organisms that have little or no DNA methylation,such as S.pombe and Drosophila.Remarkably,the Drosophila Dnmt2catalyzes a small amount of non-CG methylation early in development(Kunert et al.,2003),but the purpose of this methylation is obscure.The Dnmt2gene in S.pombe is mutated and the protein is unable to catalyze C methylation,but the gene is neverthe-less expressed for reasons that are not known(Wilkinson et al.,1995).One suggestion is that Dnmt2proteins do not methylate Cs but methylate an unusual structure that is only present in low amounts or under special condi-tions that have not been reproduced in the laboratory(Groll&Bestor,2005). Alternatively,Dnmt2proteins might serve as a structural component of chro-matin that is independent of the ability to catalyze DNA methylation(Matzke et al.,2004).
Initially,it was not clear whether RdDM requires a special DNA methyl-transferase dedicated to this process.The plant-specific CMT3,for example, was suggested as a possible candidate for carrying out RdDM(Habu et al., 2001;Matzke et al.,2001)after the chromodomain of the Drosophila histone
RNA-DIRECTED DNA METHYLATION85 acetyltransferase MOF was shown to have RNA-binding activity(Akhtar et al., 2000).However,genetic approaches in Arabidopsis have subsequently revealed that multiple,conventional DNA methyltransferases cooperate to carry out RdDM.
Reverse genetics was used to examine the role of DRM1DRM2and CMT3 in RdDM of the NOSpro.All RNA-directed de novo methylation was blocked in drm1drm2double mutants,indicating that these DNA methyltransferases catalyze the bulk of C methylation induced by RNA(Cao et al.,2003). However,DRM1DRM2are not the only enzymes required for full RNA-directed de novo methylation.Forward genetic screens of the NOSpro system recovered two mutants,rts1and rts2(RNA-mediated transcriptional silen-cing).The rts2mutant turned out to be defective in MET1.In subsequent experiments that assessed methylation in‘de novo’and‘maintenance’set-ups, MET1was shown to be required not only for preservation of CG methylation but also for full CG de novo methylation of the NOSpro(Aufsatz et al.,2004). It was concluded that the DRM1DRM2and MET1collaborate to establish de novo methylation in response to RNA signals(see Figure3.3).This might actually occur in a manner that resembles CG maintenance methylation during DNA replication,in that hemimethylated CG dinucleotides in non-replicating DNA are recognized by MET1(see Figure3.4).CMT3is usually referred to as a maintenance methyltransferase for CNG trinucleotides(Bartee et al.,2001; Lindroth et al.,2001),but it might also have some de novo activity,as has been observed in the PAI gene system(Malagnac et al.,2002).Thus,all three major types of DNA methyltransferase in Arabidopsis probably contribute to varying extents to RNA-directed de novo methylation of different sequences,probably in a manner that depends on the abundance of CG,CNG or CNN nucleotide groups.
In the context of RdDM,maintenance methylation is defined as methylation that persists after the RNA signal is withdrawn(see Figures3.1and3.3). Although there is a general consensus that asymmetrical CNN methylation is not maintained in the absence of the RNA trigger,it is still not clear how efficiently methylation in symmetrical CG and CNG nucleotide groups is preserved once the inducing RNA is removed.In one study,which used an RNA virus to induce methylation and TGS of the35S promoter,residual CG methylation was observed in uninfected progeny plants,indicating legitimate maintenance methylation in this system(Jones et al.,2001).Similarly,at least some CG methylation remains at the NOSpro after crossing out the silencing locus(Aufsatz et al.,2002a).However,all methylation is lost from the a’pro in the absence of the RNA signal(A.Matzke&T.Kanno,unpublished data). Thus,for reasons that are not yet understood,promoters vary in their ability to maintain methylation induced by RNA.One possibility is that CG and/or CNG methylation is maintained most efficiently in promoters that have a high density of the respective di-or trinucleotide.
86PLANT EPIGENETICS
The word‘reinforcement’has been used to refer to enhancement of methy-lation in cases where the RNA signal remains available after the initial de novo step(Aufsatz et al.,2002b)(see Figure3.3).Different histone-modifying activities appear to be required to reinforce CG and CNG methylation,re-spectively.For example,in the NOSpro system,the rts1mutant was found to correspond to the histone deacetylase HDA6(Aufsatz et al.,2002b).The primary methylation defect in the hda6mutant was a failure to increase CG methylation above the‘de novo’level(30–50%)initially induced by RNA. Thus,both of the rts mutants identified for the NOSpro system were impaired in some aspect of CG methylation.These results indicate that the NOSpro is silenced by CG methylation,which in turn is consistent with its constitutive nature and relatively high density of CG dinucleotides(Matzke et al.,2004). Other promoters appear to be silenced by CNG methylation,which is maintained(or reinforced)by a different histone modification(see Figure 3.3).Forward genetic screens of suppressors of hypermethylation and silen-cing of the SUP and PAI2genes identified mutants in CMT3(Bartee et al., 2001;Lindroth et al.,2001)and in the histone H3K9methyltransferase KRYPTONITE/SUVH4(KYP)(Jackson et al.,2002;Malagnac et al., 2002).The fact that CMT3and KYP/SUVH4were both identified in two independent genetic screens indicates that they frequently team up to maintain CNG methylation.The sensitivity of SUP to CNG methylation is consistent with the aforementioned sequence composition of the5’region,which is deficient in CG dinucleotides but enriched in CNGs and asymmetric CNNs. The DNA methyltransferases and histone-modifying enzymes described above are probably not dedicated exclusively to the RdDM pathway.For example,mutations in MET1(Saze et al.,2003;Vongs et al.,1993)and HDA6(Furner et al.,1998;Murfett et al.,2001;Probst et al.,2004)have been identified in screens for mutants defective in TGS(or probable TGS)that has not yet been shown to be mediated by RNA.It is therefore likely that DNA methyltransferases in Arabidopsis can respond to multiple signals for de novo methylation,of which RNA is only one.Remarkably,HDA6,which is one of 18histone deacetylases encoded in the Arabidopsis genome,is the only member of this class of proteins that has been identified in forward genetic screens for suppressors of silencing and DNA methylation.The significance of this result is not yet clear.Reverse genetic approaches have revealed require-ments for HDA1in Arabidopsis development(Tian&Chen,2001;Tian et al., 2003,2004).
3.3.2.3SNF2-like chromatin remodeling ATPases and DNA methylation
The stage of the cell cycle when RdDM occurs is not known.Clearly main-tenance methylation needs to take place during DNA replication.However,de novo methylation can potentially be catalyzed on non-replicating DNA that is packaged into chromatin,which can present a barrier to proteins and other
RNA-DIRECTED DNA METHYLATION87 regulatory factors.Therefore,it is perhaps not too surprising that forward genetic screens to recover mutants defective in DNA methylation,including RdDM in plants,have identified nucleosome-remodeling proteins of the su-crose non-fermenter(SNF2)family.These proteins use the energy of ATP to displace nucleosomes or modulate histone–DNA contacts,thus exposing DNA to regulatory factors(Lusser&Kadonaga,2003).To date,members of three subfamilies of SNF2-like proteins have been implicated in DNA methylation in either plants or mammals or in both:
(1)plant DDM1(decrease in DNA methylation)and its mammalian homolog
lymphoid-specific helicase(Lsh);
(2)mammalian ATRX(alpha-thalassemia,mental retardation,X-linked);
(3)plant DRD1(defective in RdDM).While DDM1/Lsh1and ATRX are
thought to be involved primarily in maintaining methylation at various repetitive sequences,DRD1is likely to be a factor required for RNA-directed de novo methylation.
DDM1was the first SNF2-like factor to be linked to DNA methylation.This protein was identified in a screen for mutants defective in methylation of centromeric and ribosomal DNA repeats in Arabidopsis(Vongs et al.,1993). Genomic levels of5-methylcytosine are reduced more than70%in ddm1 mutants and dramatic reductions in CG and non-CG methylation are observed in centromeric and ribosomal repeats.Although initially normal in appearance, ddm1mutants display developmental abnormalities after several generations of inbreeding(Kakutani et al.,1996).Some of these abnormalities are due to gene disruption by reactivated transposons(Miura et al.,2001)whereas others result from hypomethylation or hypermethylation of genes.Thus,ddm1 mutants are good sources of‘epimutations’,including the aforementioned hypermethylated clk epialleles of SUP(Jacobsen et al.,2000)and the hypo-methylated epialleles of FWA(Soppe et al.,2000).DDM1was eventually identified as a putative SNF2-like protein(Jeddeloh et al.,1999)and has been shown recently to promote ATP-dependent nucleosome remodeling in vitro (Brzeski&Jermanowski,2003).
DDM1is usually mentioned in connection with maintaining DNA methy-lation of transposons(Lippman et al.,2003;Singer et al.,2001)as well as sequences targeted for RdDM such as the PAI2gene(Jeddeloh et al.,1999), sense transgenes that are silenced by PTGS(Morel et al.,2000)and the NOSpro(Aufsatz et al.,2002a).Indeed,a role for DDM1in de novo methy-lation seems inconsistent with the hypermethylation of SUP in ddm1mutants (Finnegan&Kovac,2000).However,the involvement of DDM1in H3K9 methylation targeted by siRNAs(Gendrel et al.,2002)suggests that DDM1 can participate in the establishment of epigenetic modifications,at least at some loci.
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The role of DDM1in RNAi-mediated chromatin-based silencing has been studied in detail in Arabidopsis at the‘knob’,a region of interstitial hetero-chromatin on chromosome4.The chromosome4knob provides a paradigm for heterochromatin formation.It actually corresponds to one half of a seg-mental duplication that–unlike its partner–has become riddled with trans-poson insertions and related repeats,which supply the DNA foundation for creating a heterochromatic structure(Lippman et al.,2004).These repetitive sequences are preferentially targeted for DNA and histone methylation whereas adjacent single-copy genes are free of these modifications.As dem-onstrated by a microarray analysis,DNA methylation and H3K9methylation are lost from transposon sequences in the knob in ddm1mutants.The transpo-sons acquiring epigenetic modifications are sources of siRNAs,which were proposed to guide DDM1activity specifically to transposon sequences(Lipp-man et al.,2004).Consistent with this suggestion,some of the transposon short RNAs are reduced in ddm1mutants,possibly because they are normally stabilized by interactions with DDM1.Short RNAs homologous to the trans-poson-derived tandem repeats of the FWA gene have also been detected.Since DDM1contributes to the regulation of FWA expression(Soppe et al.,2000), this finding links DDM1activity to the regulation of transposons and to the regulation of genes that are adjacent to transposons via the RNAi-mediated heterochromatin pathway(Lippman et al.,2004).Many quiescent transposons that are reactivated in ddm1mutants are also reactivated in met1and hda6 mutants,suggesting that these proteins act together in a complex to maintain the transcriptionally silent state of these repetitive elements(Lippman et al., 2003)(see Figure3.3).
DDM1has a mammalian homolog,Lsh,which is important for genome-wide CG methylation in mammals(Dennis et al.,2001).In a manner similar to DDM1in plants,Lsh controls CG methylation and heterochromatin structure at pericentromeric regions comprising satellite repeats(Yan et al.,2003b).Lsh does not appear to regulate directly single-copy genes but is targeted specif-ically to repetitive elements,perhaps via small RNAs although there is no evidence as yet for this proposal(Huang et al.,2004).Because of its role in silencing repetitive elements,Lsh has been referred to as‘a guardian of heterochromatin’(Huang et al.,2004).
A second SNF2-like protein that is important for DNA methylation,at least in mammals,is ATRX.In humans,mutations in ATRX can cause ATRX syndrome(alpha-thalassemia mental retardation,X-linked),which is charac-terized by severe mental retardation,facial abnormalities,alpha-thalassemia, genital aberrations and epileptic seizures.Arabidopsis has an ATRX homolog (At1g08600),but its function has not yet been investigated.In contrast to ddm1mutants in Arabidopsis,which retain only a fraction of wild-type levels of methylation,the total amount of methylcytosine in the genome of ATRX individuals is not significantly different from normal(Gibbons et al.,2000).
肿瘤基因治疗的最新进展 王佩星 （徐州师范大学科文学院 08生物技术 088316103） 摘要：癌症是一种基因病，其发生、发展与复发均与基因的变异、缺失、畸形相关。人体细胞携带着癌基因和抑癌基因。癌症的基因治疗目前主要是用复制缺陷型载体转运抗血管生成因子、抑癌基因、前药活化基因（如ＨＳＶ－１胸腺嘧啶激酶）以及免疫刺激基因。主要抗肿瘤机制为：抑制肿瘤细胞生长、诱导肿瘤细胞凋亡、诱导抗肿瘤免疫反应、提高肿瘤细胞对化疗的敏感性、提高肿瘤细胞对放疗的敏感性、切断肿瘤细胞的营养供应。 关键词：肿瘤、基因治疗、免疫、原癌基因、抑癌基因 The latest progress of cancer gene therapy WangPeiXing (xuzhou normal university institute of biotechnology 088316103 foremen who 2008) Abstract: the cancer is a genetic disease, its occurrence, development and recurrence are associated with genetic variation, loss, deformity related. Human body cell carries oncogenes and tumor-suppressor genes. Cancer gene therapy is now primarily with copy DCF with carrier transport antiangiogenic factors, tumor-suppressor genes, before medicine activated genes (such as HSV - 1 thymine bases kinase) and immune irritancy genes. Main antitumor mechanism for: inhibiting tumor cell growth, inducing tumor cell apoptosis, inducing antineoplastic immune response, improving the sensitivity of the tumor cells to chemotherapy, radiotherapy of tumor cells to improve sensitivity, cut tumor cells to nutrition. Keywords: tumor, gene therapy, immunity, protocarcinogenic gene, tumor-suppressor genes 从本质上来讲，癌症是一种基因病，其发生、发展与复发均与基因的变异、缺失、畸形相关。人体细胞携带着癌基因和抑癌基因。正常情况下，这两种基因相互拮抗，维持协调与平衡，对细胞的生长、增殖和衰亡进行精确的调控。在遗传、环境、免疫和精神等多种内、外因素的作用下，人体的这一基因平衡被打破，从而引起细胞增殖失控，导致肿瘤发生。基因治疗的策略有基因替代、基因修复、基因添加、基因失活，目前临床使用的最主要方式是基因添加。针对肿瘤的特异性分子靶点设计肿瘤治疗方案，具有治疗特异性强、效果显著、基本不损伤正常组织的优点。这种肿瘤靶向治疗是肿瘤治疗中最有前景的方案。 1.肿瘤基因治疗的历史进展 肿瘤、艾滋病、遗传病是困扰人们的三大疾病，对肿瘤的根治是人们一直迫不及待想要实现的愿望。
基因工程的现状及发展 -标准化文件发布号：（9456-EUATWK-MWUB-WUNN-INNUL-DDQTY-KII
基因工程的现状及发展 研究背景： 迄今为止，基因工程还没有用于人体，但已在从细菌到家畜的几乎所有非人生命物体上做了实验，并取得了成功。事实上，所有用于治疗糖尿病的胰岛素都来自一种细菌，其DNA中被插入人类可产生胰岛素的基因，细菌便可自行复制胰岛素。基因工程技术使得许多植物具有了抗病虫害和抗除草剂的能力；在美国，大约有一半的大豆和四分之一的玉米都是转基因的。目前，是否该在农业中采用转基因动植物已成为人们争论的焦点：支持者认为，转基因的农产品更容易生长，也含有更多的营养（甚至药物），有助于减缓世界范围内的饥荒和疾病；而反对者则认为，在农产品中引入新的基因会产生副作用，尤其是会破坏环境。 目的意义： 如果将一种生物的 DNA中的某个遗传密码片断连接到另外一种生物的DNA 链上去，将DNA重新组织一下，就可以按照人类的愿望，设计出新的遗传物质并创造出新的生物类型。 内容摘要： 如果将一种生物的 DNA中的某个遗传密码片断连接到另外一种生物的DNA 链上去，将DNA重新组织一下，就可以按照人类的愿望，设计出新的遗传物质并创造出新的生物类型，这与过去培育生物繁殖后代的传统做法完全不同。这种做法就像技术科学的工程设计，按照人类的需要把这种生物的这个“基因”与那种生物的那个“基因”重新“施工”，“组装”成新的基因组合，创造出新的生物。这种完全按照人的意愿，由重新组装基因到新生物产生的生物科学技术，就称为“基因工程”，或者说是“遗传工程”。 基因工程在20世纪取得了很大的进展，这至少有两个有力的证明。一是转基因动植物，一是克隆技术。转基因动植物由于植入了新的基因，使得动植物具有了原先没有的全新的性状，这引起了一场农业革命。如今，转基因技术已经开始广泛应用，如抗虫西红柿、生长迅速的鲫鱼等。1997年世界十大科技突破之首是克隆羊的诞生。这只叫“多利”母绵羊是第一只通过无性繁殖产生的哺乳动物，它完全秉承了给予它细胞核的那只母羊的遗传基因。“克隆”一时间成为人们注目的焦点。尽管有着伦理和社会方面的忧虑，但生物技术的巨大进步使人类对未来的想象有了更广阔的空间。 成果展示：
[疤痕疙瘩怎么治疗]三七加醋治好疤痕疙瘩 ，所谓疤痕疙瘩是纤维瘤的一种，是与人的体质有关的疾病。那么你知道什么是疤痕疙瘩吗，疤痕疙瘩怎么治疗，疤痕疙瘩有哪些治疗的方法，下面我们一起来看看吧。疤痕疙瘩的治疗方法介绍 一、TDP远红外物理疗法 每日一次，主要针对炎性增生疤痕和烧烫伤引起的疤痕疙瘩，每次30分钟，适当延长时间可加强效果。对软化疤痕，促进创伤恢复，消除炎症有很好的作用。 二、超音导入法 超音波能使胶原蛋白纤维素分散，对疤痕组织有一定的软化作用，每日一次，每次8-15分钟，15次为一疗程，促进药物吸收。 三、指压按摩 热疗后逆时针稍用力按摩10分钟左右。 四、加压疗法 一般选用弹力套，必须在病人身上量身定做。 本法适用于疤痕面积大，不适宜局部药物治疗者，对预防疤痕增生、及增生期疤痕有效，对减退期及成熟期疤痕无效。 五、内治疗法 在疤痕治疗中应遵循辩证统一的原则，以外治为主，有症状的顽固性疤痕、大面积疤痕及青壮年疤痕患者最好配合内治法。 在日常生活中，对于怎样治疗疤痕疙瘩，我们还必须注意以下几个方面。 一、生活饮食一定要清淡，多吃水果蔬菜类食物，尽量少食辛辣、海鲜、酒等刺激性食物。 二、疤痕疙瘩痛痒症状严重时，可用手拍打或采用冰敷的方法，不要用手抓挠。这些都会加重疤痕增生。 三、一些疤痕部位没有汗毛，不容易排汗，所以建议患者穿全棉的衣服，降低对疤痕的刺激。 四、切记不要用非医嘱药或激光治疗，以免刺激皮肤，加速疤痕疙瘩的增生。 五、如疤痕疙瘩面积不大、已经停止生长，没有痛痒的症状，或是症状不明显，不影响您本人的正常工作和生活，不建议治疗。疤痕疙瘩预防措施 疤痕疙瘩是创伤、外伤的一种重要的并发症。烧烫伤、外伤、创伤、痤疮、打耳孔、打预防针等都可以形成不同程度的疤痕疙瘩和疤痕增生。 疤痕疙瘩的预防措施主要在于疤痕形成前、形成间的尚未成熟阶段。主要目的就是要尽量避免可能产生疤痕增生的因素，减少疤痕的生长、预防疤痕对机体造成的各种畸形及功能障碍。 因此有效预防疤痕疙瘩，就需要平时注意防止外伤、烧烫伤、打耳孔、纹眉线等，尤其是免疫功能差的部位，如胸前、肩背等处，以免损失真皮。如果疤痕严重时，要注意减少对患处的机械、化学、热力的刺激，避免反复牵拉、摩擦溃破及感染的发生。另外，平时要注意饮食，因为某些食物能刺激疤痕疙瘩增生得更快，而有些食物可以让疤痕疙瘩趋于稳定状态。 饮食提示 1、勿偏食 中医认为药食同源。食物也具有寒热温凉四性和酸苦甘辛咸五味。如有偏食则会导致身体摄纳的营养不平衡，不利于瘢痕的消退。
基因治疗的现状与展望 朱双喜 在生命进化的漫长历程中，生物体通过基因突变以适应环境，基因突变是生物进化的基础。同时，不利的突变会造成细胞形态和功能的异常，导致疾病，甚至机体的死亡。人体某些疾病的发生与基因的核苷酸序列变化有关，那么从校正核苷酸序列着手来治疗疾病的设想也就顺理成章了。基因治疗是向靶细胞引入正常的或野生型基因，纠正和补偿致病基因产生的缺陷从而达到治疗疾病的目的。．Anderson于80年代初首先阐述了基因治疗的前景；1990年美国成功地进行了ADA(腺苷脱氨酶)缺陷患儿的人体基因治疗；1991年我国首例基因治疗B型血友病也获得了成功。目前，基因治疗已从遗传病扩展到肿瘤、心血管疾病、神经系统疾病、传染病，包括AIDS病等领域。它依然存在例如缺少高效的传递系统、缺少持续稳定的表达和寄主产生免疫反应等一些问题。但随着人类基因组计划的实施、大批新基因的发现以及新技术的发展，治疗范围将大大拓宽，从而给人类健康事业带来深远的影响。一、基因治疗的前提 在基因治疗成为一种普通的医疗手段之前必须首先明确两个前提。 1．1 基因治疗需要有清晰定义的靶组织通常以疾病类型来选择进行基因治疗的细胞，例如在肺部系统纤维疾病的临床治疗中选择肺作为靶器官，使用呼吸道气雾剂法，可使含有补偿缺陷基因的DNA直接传递到肺中；治疗类似血友病的凝血因子疾病需要在血浆里含有达到治疗水平的凝血蛋白，这种蛋白可以由肌肉、活细胞、成纤维细胞或甚至血细胞提供，于是就可有多种接受基因治疗的靶组织。此外，靶组织的最终选择还必须考虑基因传递的效率、表达蛋白变性、机体免疫状态、可行性和治疗费用等因素。 1．2 究竞要往靶组织内传递多少治疗基因B型血友病的病因是缺乏一种称为第九因子的凝血蛋白，然而病人只需正常水平5％的这种蛋白，其生存机会就能提高，假设经治疗后的细胞能稳定表达这种蛋白，那么需要传递基因给人体全部1013个细胞中的5xlO11细胞；然而相对于大脑来说，只需几百个细胞被基因转染，神经性疾病的患者就可减轻痛苦；如果考虑对成血干细胞(或生殖细胞)进行基因转染，治疗几个细胞将会对其数以百万计的子代产生影响，所起的负作用也同样如此。 二、基因治疗的方法 基因治疗的应用有两种途径：(1)把一个健康基因拷贝插入靶细胞以补偿缺陷基因；(2)引进经过改造的基因以赋予细胞新的特性。最常用的技术有：(a)体外处理(ex vivo)疗法～将有基因缺陷的细胞取出，引入正常基因拷贝后再送回体内；(b)原位疗法，使用载体将目的基因直接导入靶组织。(c)体内疗法(in vivo)，将基因载体注入血液，定向寻找靶细胞并将遗传信息安全有效地导入。 基因治疗载体可分为病毒型和非病毒型[4]两类。病毒型载体包括：逆转录病毒、腺病毒、腺相关病毒和疱疹病毒，目前最有效的方法是使用经过改造的、具有穿膜特性的病毒作为载体，定向地将目的基因导入细胞。然而由于人体自身具有抗病毒的免疫系统，使用病毒载体作为媒介来传递DNA时就不得不面对宿主的免疫反应。非病毒型载体包 括：脂质体、裸露DNA和DNA包装颗粒，范围从裸DNA显微注射，电激法、基因枪技术等各类物理学方法到聚阳离子赖氨酸或阳离子脂质体。 三、基因治疗面临的问题 缺乏某种单基因产物而患病的病人，一旦获得一个野生型的基因拷贝并能正常表达，就有被治愈的可能。基因治疗亟待解决的问题是目的基因的定向表达，目的基因导入靶细胞是定向表达的基本条件。目前，必须优先发展有更强适用性和灵活性的能准确调控转导基因表达的基因传递系统，即发展一种理想的“载体”(能帮助新的基因"潜入")，人体细胞的特殊
Big Genomic Data in Bioinformatics Cloud Abstract The achievement of Human Genome project has led to the proliferation of genomic sequencing data. This along with the next generation sequencing has helped to reduce the cost of sequencing, which has further increased the demand of analysis of this large genomic data. This data set and its processing has aided medical researches. Thus, we require expertise to deal with biological big data. The concept of cloud computing and big data technologies such as the Apache Hadoop project, are hereby needed to store, handle and analyse this data. Because, these technologies provide distributed and parallelized data processing and are efficient to analyse even petabyte (PB) scale data sets. However, there are some demerits too which may include need of larger time to transfer data and lesser network bandwidth, majorly. 人类基因组计划的实现导致基因组测序数据的增殖。这与下一代测序一起有助于降低测序的成本，这进一步增加了对这种大基因组数据的分析的需求。该数据集及其处理有助于医学研究。 因此，我们需要专门知识来处理生物大数据。因此，需要云计算和大数据技术（例如Apache Hadoop项目）的概念来存储，处理和分析这些数据。因为，这些技术提供分布式和并行化的数据处理，并且能够有效地分析甚至PB级的数据集。然而，也有一些缺点，可能包括需要更大的时间来传输数据和更小的网络带宽，主要。 Introduction The introduction of next generation sequencing has given unrivalled levels of sequence data. So, the modern biology is incurring challenges in the field of data management and analysis. A single human's DNA comprises around 3 billion base pairs (bp) representing approximately 100 gigabytes (GB) of data. Bioinformatics is encountering difficulty in storage and analysis of such data. Moore's Law infers that computers double in speed and half in size every 18 months. And reports say that the biological data will accumulate at even faster pace [1]. Sequencing a human genome has decreased in cost from $1 million in 2007 to $1 thousand in 2012. With this falling cost of sequencing and after the completion of the Human Genome project in 2003, inundate of biological sequence data was generated. Sequencing and cataloguing genetic information has increased many folds (as can be observed from the GenBank database of NCBI). Various medical research institutes like the National Cancer Institute are continuously targeting on sequencing of a million genomes for the understanding of biological pathways and genomic variations to predict the cause of the disease. Given, the whole genome of a tumour and a matching normal tissue sample consumes 0.1 T B of compressed data, then one million genomes will require 0.1 million TB, i.e. 103 PB (petabyte) [2]. The explosion of Biology's data (the scale of the data exceeds a single machine) has made it more expensive to store, process and analyse compared to its generation. This has stimulated the use of cloud to avoid large capital infrastructure and maintenance costs. In fact, it needs deviation from the common structured data (row-column organisation) to a semi-structured or unstructured data. And there is a need to develop applications that execute in parallel on distributed data sets. With the effective use of big data in the healthcare sector, a
治疗疤痕最好的办法 说到疤痕很多人都陌生，因为我们的身上有很多大大小小的疤痕，这种现象是因为我们的身体出现了伤口，而且伤到了真皮层，这样就会导致疤痕的出现，会让我们的身体美观大打折扣，所以我们一定要及时的解决这样的情况。在我们的生活中治疗疤痕的偏方有很多种，下面我们一起了解下。 疤痕如果出现在隐秘的地方的话，那么还不会让我们的形象受到影响，但是如果出现在非常暴露的地方的话，那么就会让我们的形象受到影响，还会让我们产生自卑的心理，所以我们一定要及时解决，让我们可以早日拥有完整的皮肤。 1、把三七磨成粉末，加上适量的醋，对瘢痕疙瘩的治疗也有一定的效果。三七本身就有活血化瘀的功效，通过活血化瘀使患处气血畅通，以促使疤痕变平变软，能够消除痛痒、软化瘢痕疙瘩，长期外敷能祛除瘢痕疙瘩。
2、该处方由六种药物组成：分别是白附子、辛夷、细辛、鸡屎白。白附子具有解毒散结止痛，辛夷、细辛皆能祛风通窍，鸡屎白能利水泄热，这四味药一起使用可疏散风热，散结消肿有一定的祛除疤痕的效果。 3、还有一种效果比较好的小偏方，它是由鸡屎白、鹰粪白、芍药、白蔹、衣中白鱼、白蜂六味药物组成的。鸡屎白能利水泄热，祛风解毒;白蔹能消肿生肌，清热解毒;芍药能活血补血，润肤红颜;鹰粪白能滋养容颜，滑丽皮肤;衣中白鱼能祛风解毒，因此受到了大家的青睐，但是同时提醒广大患者朋友，每个人的体质不同，在入药的时候应当咨询专家，科学使用偏方，以免造成危害。 针对治疗疤痕最好的办法的问题就讲解到这里，希望对您起到帮助，通过以上的介绍我们可以得知，治疗疤痕的偏方非常多，但是我们在选择的时候一定要详细的了解，在专业人士的指导下使用，以保证给我们的皮肤不受到疤痕的影响。
基因组生物信息学 Introduction to Genomics and Bioinformatics 基因组生物信息学是在人类基因组计划完成之后出现的热点研究领域。本课程是一门创新的课程，通过理论学习与实际操作演示介绍基因组和生物信息学中前沿与常用的知识和工具，使得学生在理论与技能两方面得到培养。课程内容新颖，紧跟国际最新研究进展与热点，结合应用实例介绍实用的技能。课程开设目的是让选课学生了解基因组生物信息学的基本概念、掌握基本工具；认识基因组学与日常生活的密切关系，和基因组技术个体化对社会和人们生活方式的深远影响；同时培养兴趣，为该领域吸引跨学科人才。课程内容包括：基因组学与生物信息学简介，基因组基本理论，人类基因组计划的历史与未来，基因组测序方法与进展，基因组序列注释方法，转录组学简介，比较基因组与进化基因组学简介等。教学团队的几位教师都是这一领域的专家，其中赵国屏院士是微生物基因组学方面的专家，曾经在抗击SARS的研究中作出突出贡献；周雁教授主持与参与了多个大型基因组研究计划，包括人、水稻、血吸虫等，在基因组与生物信息学方面有着丰富的理论与实践经验。 赵国屏，教授，中科院院士复旦大学 gpzhao@https://www.doczj.com/doc/6a11271766.html, 现任国家人类基因组南方研究中心执行主任，生物芯片上 海国家工程中心主任，复旦大学微生物学与微生物工程系 主任，中国微生物学会理事长。主要研究方向为微生物基 因组学和生物信息学，进化，代谢调节，合成生物学。 周雁，副教授复旦大学 zhouy@https://www.doczj.com/doc/6a11271766.html, 复旦大学生命科学学院副教授，上海市生物信息学会理事。 主要研究方向为病源与宿主在基因组和表达组水平的相互 作用，及在此过程中相关基因家族及其功能的进化规律。教师风采
基因治疗的发展及其应用 【摘要】基因治疗一种很有发展前途的高新技术。基因治疗有望成为治疗遗传病、肿瘤、心血管病、病毒感染及其它难治性疾病的有效手段，本文通过国内外相关文献的分析，从基因治疗(基因治疗的现状、肿瘤的基因治疗)、基因预防、基因治疗技术、基因治疗存在的问题和未来发展等进行综述。 【关键词】基因治疗;基因预防;基因治疗技术;现状;问题和未来发展 人类的疾病是由于其本身的基因的核苷酸发生变化有关。近年来，基因治疗作为一种安全的、新的疾病治疗手段，在一定程度上取得了重大进展。 1 基因治疗 基因治疗(Genethrapy)是向靶细胞引入正常有功能的基因，以纠正或补偿致病基因所产生的缺陷，从而达到治疗疾病的目的，通常包括基因置换、基因修正、基因修饰、基因失活等。简而言之，基因治疗是指通过基因水平的操纵而达到治疗或预防疾病的疗法。 1.1 基因治疗的现状 生物医学的深入研究表明，人类的各种疾病都直接或间接与基因有关[1]。因此，可认为人类的一切疾病都是“基因病”。故人类疾病可分为三大类。一类是单基因病。这类疾病只需一个基因缺陷即可发生，如腺苷脱氨基酶(ADA)缺陷症。二是多基因病。此类疾病的病因大多比较复杂，不但涉及各个基因，往往还与环境因素(包括自然环境、社会环境、生活方式等)有关。基因缺陷和疾病表型都具有明显的多样性。Ⅰ型糖尿病、肿瘤、心血管疾病等皆属此类。三是获得性基因病。此乃病原微生物入侵所致，如艾滋病、乙型肝炎等。因此，理论上，人类所有的疾病都可采用基因治疗。 1.2 肿瘤的基因治疗 目前治疗癌症的基因疗法种类颇多，主要集中在免疫基因治疗、药物敏感性基因治疗、肿瘤抑制基因治疗治疗三个方面。 1.2.1 免疫基因治疗 常用方法有：①细胞因子基因治疗：将某些细胞因子基因如IL 2、IL 4、IL 6、B7 1，GM CSF等转染肿瘤细胞后，增强机体对肿瘤细胞的免疫反应。②肿瘤抗原基因免疫治疗：将某些肿瘤抗原基因如MHC基因等转染肿瘤细胞，增强肿瘤细胞免疫原性。②反义基因治疗：应用反义核酸在转录和翻译水平，通过碱基互补原则封闭某些异常基因的表达，反义核酸被称为信息药物[3]。④用抗体抑制癌基因的产物杀灭肿瘤细胞。
基因治疗发展趋势与中国现状思考 日期：2015-03-04 09:52:12 作者：协和医院戴毅 对于罹患肌营养不良等单基因病的患者和家庭来说，修复基因缺陷是治疗的希望。基因治疗也就成为众望所归。 应该说，近年来随着生物科学、分子技术的进步，基因治疗正在加速发展。2012年10月，欧盟批准了西方发达国家第一个基因治疗药物——uniQure公司的Glybera，其利用经过改造的AAV1病毒载体将LPL（Lipoprotein Lipase）基因导入患者肌肉细胞内，产生脂蛋白脂肪酶，纠正患者基因缺陷导致的酶缺失，进而消除反复发作胰腺炎等临床症状。经过两年的运作和筹备，本药有望在几个月内在欧盟上市（首先选在德国），初步定价111万欧元（约800万人民币），当然上市前还要和德国政府医疗保险部门讨价还价。对于整个基因治疗领域，这是一个具有里程碑意义的事件。 这里要提一下，在所有正式报道中都会说明，这是在Western world中第一个上市的基因治疗制剂。之所以不是全球第一，因为早在2004年中国就批准上市了全球第一个基因治疗药物——今又生（重组人P53腺病毒注射液），用于晚期鼻咽癌患者配合放疗的联合治疗。但当时支持其上市的临床试验数据非常有限，一直被广为质疑。该药上市后的临床应用也很有限，2008年因生产流程问题，SFDA吊销了该公司的GMP生产许可。此外，早在2003年，国内rAAV2-hFIX 治疗血友病B的I期临床试验就被SFDA批准，可惜其后并未有后续结果。 从世界范围来讲，基因治疗（特别是AAV介导的基因治疗）无疑已进入高速发展的快车道。通过20余年的前期积累，其安全性和有效性已获得医药监管部门和医药巨头的广泛认可（AAV用于最脆弱易感的SMA新生儿的临床试验已在美国开展，详见此前文章）。世界制药巨头纷纷投入到基因治疗药物上市前最后阶段的推动中，诺华、辉瑞、赛诺菲、拜耳、百特都通过收购基因治疗公司或与之合作进入这一领域，极大的推动了III期临床试验的开展。基因治疗公司已成为Nasdaq最为耀眼的明星。除了罕见病，制药巨头更看重的是，在精准医学时代，对于常见病的治疗也必须将患者细化，按照罕见病的模式，将更为精准的针对性治疗，如基因治疗，用于今后的治疗。 在国外一片风起云涌的情况下，国内的基因治疗显得很沉寂。近10年，国内没有任何新的基因治疗临床试验获批实施，也没有基因治疗领域的国际多中心临床试验。究其原因，国内的科研转化仍然比较落后，而新兴的基因治疗领域又是烧钱大户，前期投入非常巨大，稍有不慎就可能全军覆没，就连这一领域的翘楚Introgen公司都因为FDA未认可其P53基因治疗III期临床试验结果，导致其不得不申请破产保护。因此国内基因治疗产业由于缺少资本注入，起步非常困难。 另一方面，国内患者参与国外基因治疗临床试验也存在困难，主要原因如下：、我国CFDA明确规定，国外新药不能在国内做I期和II期临床试验。只有在国外完成I期和II期临床试验后，才可以申请在国内进行III期临床试验或多中心III期临床试验。国家如此规定是为了避免国人成为外国制药企业的“小白鼠”（印度由于相关管制较松，而成本大大低于发达国家。有大量西方国家的新药在印度进行I期、II期临床试验，甚至出现违背伦理学原则的情况，经常见诸报端）。但相应的，也提高了新药进入我国的门槛。 2、中国是发展中大国，医疗保险和居民收入水平相较发达国家仍有较大差
找了一些中药治疗疤痕的偏方，有需要的朋友进来看看 2009-04-14 上午 10:41 下面是找的药方，用前先咨询一下中医 1夏氏遵前人观点主张瘢痕主要为淤血阻滞，治以水蛭活血汤：水蛭9g～15g，桃仁、红花、制乳香、制没药、三棱、莪术、伸筋草、炙山甲、威灵仙，病在上肢加桑枝、桂枝，病在下肢加川牛膝、麻木加全蝎、蜈蚣、并强调加水蛭为方中主药，缺之难以获效 2史氏等对瘢痕进行辨证分型，分为实热型、虚实错杂型、溃脓型、并自拟“消积排通汤”消积排浊，药用白芷、甲珠、雷丸、寸冬、元胡、桃仁、红花、榔片、荆芥等。。 3赵氏以内服消瘢汤(丹参30g～60g，陈皮、半夏、炙山甲、皂角刺、白芥子各10g、川芎、红花、羌活、独活各20g，，蔓荆子、苍耳子各6g)加减，共治疗9例，治愈5例，显效3例，有效1例。 4史氏等对瘢痕的发病机理提出“实证是基本，虚证是其标”的新理论，运用消积排通汤(白芷、甲珠、延胡索、桃仁、红花、荆芥等)内服， 5刘氏认为本病治疗应活血化瘀、清热解毒、散结消瘢，方选《医宗金鉴》凉血四物汤加味：当归、生地、赤芍、金银花、川芎、红花、陈皮、赤苓、黄芩、丹皮、三棱、莪术、大黄、桔梗、甘草。 6 生肌化瘀方：黄芪45 g，太子参30 g，白术15 g，生地黄15 g，丹参30 g，水蛭9 g，桃仁12 g，川芎12 g。（生肌方：黄芪45 g，太子参30 g，白术15 g，生地黄15 g。化瘀方：丹参30 g，水蛭9 g，桃仁1 2 g，川芎12 g），这个实验里面的化淤方效果最好,里面的药物也比较常见,所以值得推荐.川芎,我自己买了一些,准备做实验用.丹参是个不错的药物,这两味中药的提取物,在心血管科室里面使用率很高,如果硬是要用西医化的理论解释,就是抗凝血和抗氧化.水蛭也是很强的抗凝血药物,桃仁在抗凝和抗纤维化有不错的效果,共同作用,软化疤痕,防止纤维交连变硬.西医方面,外用的药膏以肝素钠为代表,就是个典型的抗凝血药物.还有硅类防止纤维增生.所以治疗疤痕,中西医大部分还是共通的,就是抗凝,抗氧化,防止纤维过度生长.个人认为,喝这个药的最好时机就是疤痕已经长稳固,而正在慢慢改建成与周围组织相似的过程中.而不再有变化的旧疤痕,效果不会很明显,细胞的改建活动已经不太活跃了. 7五灵脂丸:组成：五灵脂1500克。功效：活血破淤，软坚化滞。主治：瘢痕疙瘩。用法：研细末，炼蜜为丸，每丸3克。每次半丸至一丸半，日2次，温开水送下。 8茄子消瘢剂: 颜面或身体受伤青肿，可用老黄茄子极大者，切片如一指厚，新瓦焙研为未。欲卧时温酒调服7．5克。古人称应用此方，“一夜消尽且无痕迹”。出处：《胜金方》 9瘢痕疙瘩:香附9克，柴胡9克，川芎9克，熟地12克，当归12克，赤芍9克，穿山甲9克，夏枯草15克，梨树根45克。功效：调和营卫，舒畅气血。主治：瘢痕疙瘩，食道粘连，食道憩室。用法：将穿山甲、梨树根用清水浸泡30分钟，再入余药，煎煮30分钟，每剂煎2次，将2次煎液混合。每日1剂，早、晚各服1次，食道粘连者可日服3-5次
基因治疗的发展现状和存在风险分析 发表时间：2019-06-24T10:40:47.343Z 来源：《成功》2019年第1期作者：傅思博 [导读] 随着生物科学的发展，科学家们逐渐解开了生命的密码——基因，作为遗传信息的DNA片段，人们发现可以通过改善或改变基因的排列组合，或通过外源性基因的植入，可以解决非常多的遗传疾病，乃至于改善后代生育的质量。但同时外源性基因侵入的风险和伦理问题也是基因工程面临的主要问题。 傅思博 西安市铁一中陕西西安 710016 【摘要】随着生物科学的发展，科学家们逐渐解开了生命的密码——基因，作为遗传信息的DNA片段，人们发现可以通过改善或改变基因的排列组合，或通过外源性基因的植入，可以解决非常多的遗传疾病，乃至于改善后代生育的质量。但同时外源性基因侵入的风险和伦理问题也是基因工程面临的主要问题。 【关键词】基因；基因工程；基因治疗；风险分析 一、基因与基因工程概述 （一）基因就是带有遗传信息的DNA片段，它是控制生物性状的基本遗传单位 基因（遗传因子）是产生一条多肽链或功能RNA所需的全部核苷酸序列。基因支持着生命的基本构造和性能。储存着生命的种族、血型、孕育、生长、凋亡等过程的全部信息。环境和遗传的互相依赖，演绎着生命的繁衍、细胞分裂和蛋白质合成等重要生理过程。生物体的生、长、衰、病、老、死等一切生命现象都与基因有关。它也是决定生命健康的内在因素。因此，基因具有双重属性：物质性（存在方式）和信息性（根本属性）。 基因有两个基本特点：一是能忠实地复制自己，以保持生物的基本特征；二是在繁衍后代上，基因能够突变或变异，当受精卵或母体受到环境或遗传的影响，后代的基因组会发生有害缺陷或突变。 （二）基因工程的基本定义 基因工程又称基因拼接技术和DNA重组技术，它是在分子水平上对基因进行操作的复杂技术，是将外源基因通过体外重组后导入受体细胞内，使这个基因能在受体细胞内复制、转录、翻译、表达。也就是说，它是将一种生物体的基因与载体在体外进行拼接重组，然后转入另一种生物体内，使之按照人们的意愿稳定遗传并表达出新产物或新性状。基因工程是在分子生物学和分子遗传学综合发展的基础上诞生的一门崭新的生物技术科学。它与细胞工程、酶工程、蛋白质工程和微生物工程共同组成了生物工程。 实施基因工程一般包括四个操作步骤：一是提取目的基因；二是目的基因与运载体结合；三是将目的基因导入受体细胞内；四是目的基因的检测和表达。 二、基因治疗的发展现状 随着人类对基因研究的不断深入，发现许多疾病是由于基因结构与功能发生改变所引起的。科学家将不仅能发现有缺陷的基因，而且还能掌握如何进行对基因诊断、修复、治疗和预防，这是生物技术发展的前沿。这项成果将给人类的健康和生活带来不可估量的福音。 （一）基因治疗的原理 基因治疗是指以改变人类遗传病物质为基础的生物医学治疗,即通过一定方式将人正常或野生型基因或有治疗作用的DNA顺序导入人体靶细胞,以矫正或置换致病基因的治疗方法[1]。已发现的遗传病有6500多种，其中由单基因缺陷引起的就有约3000多种。因此，遗传病是基因治疗的主要对象。第一例基因治疗是美国在1990年进行的。当时，两个4岁和9岁的小女孩由于体内腺苷脱氨酶缺乏而患了严重的联合免疫缺陷症。科学家对她们进行了基因治疗并取得了成功。这一开创性的工作标志着基因治疗已经从实验研究过渡到临床实验。1991年，我国首例B型血友病的基因治疗临床实验也获得了成功。 （二）基因治疗的最新进展 基因治疗的最新进展是即将用基因枪技术用于治疗。其方法是将特定DNA用改进的基因枪技术导入小鼠的肌肉、肝脏、脾、肠道和皮肤获得成功的表达。这一成功预示着人们未来可能利用基因枪传送药物到人体内的特定部位，以取代传统的接种疫苗，并用基因枪技术来治疗遗传病。 按照遗传基本原理，如果某些基因能帮助父母生存和繁殖，父母就会把这些基因传给后代。但一些研究表明，真实情况要复杂得多：基因可以被关闭或沉默，以应对环境或其他因素，这些变化有时也能从一代传到下一代。美国马里兰大学遗传学家提出了一种特殊机制，父母通过这种机制可以把沉默基因遗传给后代，而且这种沉默可以保持25代以上。这一发现可能改变人们对动物进化的理解，有助于将来设计广泛的遗传疾病疗法。 所以，据此我们可以设想，第一、在孕期进行产前诊断，检测出遗传病基因后提取胚胎内细胞团，通过基因重组将人工编辑的dsRNA 基因导入内细胞团中，使遗传病的基因暂时性无法正常表达后进行基因编辑与剪辑替换。第二、暂时延缓遗传类疾病的病情恶化，延缓发病时间，为医生和患者争取更多治疗时间。如果实验疗效得到进一步确证的话，就有可能将胎儿基因疗法扩大到其它遗传病，以防止出生患遗传病症的新生儿，从而从根本上提高后代的健康水平。 三、基因治疗的风险分析 （一）现阶段基因治疗的技术风险 基因治疗的技术风险是指，由于技术本身的不成熟而导致患者，受试者的身心伤害。它包括疾病的种类，致病概率的大小和风险的严重程度等三个方面。[2] 在基因临床试验中，技术风险表现在三个方面：1.如何选择有效的治疗基因；2.如何构建安全载体，病毒载体效率较高，但却有潜在的危险性；3.如何定向导入靶细胞，并获得高表达。 （二）现阶段基因治疗的伦理风险 基因工程同其他科学技术成果一样也是一把双刃剑：它一方面给人类带来了巨大的福祉,但同时也产生了巨大的伦理风险。基因治疗技术的快速发展使我们不得不认真思考“人是什么”这个问题，如果人类可以改良基因，那么人类基因的权利何在？因此,由基因的特殊性所引
一、名词解释(31个) 1.生物信息学:广义：应用信息科学的方法和技术，研究生物体系和生物过程中信 息的存贮、信息的内涵和信息的传递，研究和分析生物体细胞、组织、器官的生理、病理、药理过程中的各种生物信息，或者也可以说成是生命科学中的信息科学。狭义：应用信息科学的理论、方法和技术，管理、分析和利用生物分子数据。 2.二级数据库：对原始生物分子数据进行整理、分类的结果，是在一级数据库、实验 数据和理论分析的基础上针对特定的应用目标而建立的。 3.多序列比对：研究的是多个序列的共性。序列的多重比对可用来搜索基因组序列的 功能区域，也可用于研究一组蛋白质之间的进化关系。 4.系统发育分析：是研究物种进化和系统分类的一种方法，其常用一种类似树状分支 的图形来概括各种（类）生物之间的亲缘关系，这种树状分支的图形称为系统发育树。 5.直系同源：如果由于进化压力来维持特定模体的话，模体中的组成蛋白应该是进化 保守的并且在其他物种中具有直系同源性。 指的是不同物种之间的同源性，例如蛋白质的同源性，DNA序列的同源性。（来自百度） 6.旁系（并系）同源：是那些在一定物种中的来源于基因复制的蛋白，可能会进化出 新的与原来有关的功能。用来描述在同一物种内由于基因复制而分离的同源基因。 （来自百度） 7.FASTA序列格式：将一个DNA或者蛋白质序列表示为一个带有一些标记的核苷酸或 氨基酸字符串。 8.开放阅读框（ORF）：是结构基因的正常核苷酸序列，从起始密码子到终止密码子 的阅读框可编码完整的多肽链，其间不存在使翻译中断的终止密码子。（来自百度）9.结构域：大分子蛋白质的三级结构常可分割成一个或数个球状或纤维状的区域，折 叠得较为紧密，各行其功能，称为结构域。 10.空位罚分：序列比对分析时为了反映核酸或氨基酸的插入或缺失等而插入空位并进 行罚分，以控制空位插入的合理性。（来自百度） 11.表达序列标签：通过从cDNA文库中随机挑选的克隆进行测序所获得的部分cDNA的 3’或5’端序列。（来自文献） 12.Gene Ontology 协会： 13.HMM 隐马尔可夫模型：将核苷酸序列看成一个随机序列，DNA序列的编码部分与非 编码部分在核苷酸的选用频率上对应着不同的Markov模型。 14.一级数据库：数据库中的数据直接来源于实验获得的原始数据，只经过简单的归类 整理和注释 15.序列一致性：指同源DNA顺序的同一碱基位置的相同的碱基成员, 或者蛋白质的同 一氨基酸位置的相同的氨基酸成员, 可用百分比表示。 16.序列相似性：指同源蛋白质的氨基酸序列中一致性氨基酸和可取代氨基酸所占的比 例。 17.Blastn：是核酸序列到核酸库中的一种查询。库中存在的每条已知序列都将同所查 序列作一对一地核酸序列比对。（来自百度） 18.Blastp：是蛋白序列到蛋白库中的一种查询。库中存在的每条已知序列将逐一地同 每条所查序列作一对一的序列比对。（来自百度）
当前基因治疗的现状和一般性技术路线 基因治疗是当前医学领域研究的热点和前沿问题，本文论述了基因治疗的现状和一般性技术路线，供相关领域的研究人员和一般科学工作者参考。 标签：基因治疗CGT CAR-T 基因治疗目前作为一种非常规疗法，对于彻底治疗一些具有罕见破坏性遗传病，如交界性大疱性表皮松解症（junctional EB，JEB），和一些罕见的进行性神经肌肉功能损坏性遗传病，如Ι型脊髓性肌萎缩症（SMAΙ），和一些常见的延迟发病的神经功能退行性疾病，如帕金森病（Parkinson’s disease，PD），还有特定的病毒感染性疾病（如艾滋病，AIDS）和某些种类的恶性肿瘤（如急性淋巴白血病，ALL），都已经表现出了光明的前景。细胞和基因治疗领域（Cell and Gene Therapy，CGT）也是当今生物医学研究的重要的前沿和热点课题。 一、当前基因治疗的现状 当前基因治疗技术还不是很成熟的治疗方式，总体上还处在实验探索的阶段，还具有一定的风险性，其安全性和有效性还需要有很大的提高。往往是在没有很好的常规治疗方式前提下，对于一些危害性较大的疾病的一种试验性治疗。对于不同的疾病，其治疗效果往往相差很大；不同的机构对于同一种疾病，其治疗的过程也有较大差异。另外，基因治疗前期要投入大量人力、物力和时间，所面对的又是小范围内的患者，所以导致了其高昂的治疗费用。 二、当前基因治疗的一般性技术路线 当前基因治疗的主要有三种情况： 第一种，外源基因基因直接导入法 虽然说是基因直接导入，却不是直接将基因片段导入靶器官，因为直接导入，基因无法整合到靶细胞基因组里面，也就得不到表达。常用的方法就是将腺相关病毒（Adeno-Associated Virus，AA V）作为载体，然后导入靶器官。为了实现更高的目的蛋白质表达量和降低免疫毒性，往往还需要对载体进行改造，对目的基因进行密码子优化。根据《自然·通讯》报道，英法合作团队以狗为动物试验模型地开发出了较为成功的针对杜氏肌营养不良症（DMD）的基因疗法。DMD患者均为男性，患者在几岁时就会表现出肌无力症状，后来会恶化到无法自己行走，随后各种肌无力问题相继出现。一种抗肌萎缩蛋白有关的基因变异影响了抗肌萎缩蛋白的生成，进而影响肌肉的功能，导致了DMD。研究小组将抗肌萎缩蛋白基因进行了“压缩”，制作出功能性缩微版抗肌萎缩蛋白，并成功利用AA V载入12只DMD狗的体内。他们仅对这些狗进行注射治疗了一次，两年来这些狗的病情被成功抑制，不但临床症状稳定，肌肉功能还得到了很大程度的改善。
1夏氏遵前人观点主张瘢痕主要为淤血阻滞，治以水蛭活血汤：水蛭9g～15g，桃仁、红花、制乳香、制没药、三棱、莪术、伸筋草、炙山甲、威灵仙，病在上肢加桑枝、桂枝，病在下肢加川牛膝、麻木加全蝎、蜈蚣、并强调加水蛭为方中主药，缺之难以获效 2史氏等对瘢痕进行辨证分型，分为实热型、虚实错杂型、溃脓型、并自拟“消积排通汤”消积排浊，药用白芷、甲珠、雷丸、寸冬、元胡、桃仁、红花、榔片、荆芥等。。 3赵氏以内服消瘢汤(丹参30g～60g，陈皮、半夏、炙山甲、皂角刺、白芥子各10g、川芎、红花、羌活、独活各20g，蔓荆子、苍耳子各6g)加减，共治疗9例，治愈5例，显效3例，有效1例。 4史氏等对瘢痕的发病机理提出“实证是基本，虚证是其标”的新理论，运用消积排通汤(白芷、甲珠、延胡索、桃仁、红花、荆芥等)内服， 5刘氏认为本病治疗应活血化瘀、清热解毒、散结消瘢，方选《医宗金鉴》凉血四物汤加味：当归、生地、赤芍、金银花、川芎、红花、陈皮、赤苓、黄芩、丹皮、三棱、莪术、大黄、桔梗、甘草。 6 生肌化瘀方：黄芪45 g，太子参30 g，白术15 g，生地黄15 g，丹参30 g，水蛭9 g，桃仁12 g，川芎12 g。（生肌方：黄芪45 g，太子参30 g，白术15 g，生地黄15 g。化瘀方：丹参30 g，水蛭9 g，桃仁1 2 g，川芎12 g），这个实验里面的化淤方效果最好,里面的药物也比较常见,所以值得推荐.川芎,我自己买了一些,准备做实验用.丹参是个不错的药物,这两味中药的提取物,在心血管科室里面使用率很高,如果硬是要用西医化的理论解释,就是抗凝血和抗氧化.水蛭也是很强的抗凝血药物,桃仁在抗凝和抗纤维化有不错的效果,共同作用,软化疤痕,防止纤维交连变硬.西医方面,外用的药膏以肝素钠为代表,就是个典型的抗凝血药物.还有硅类防止纤维增生.所以治疗疤痕,中西医大部分还是共通的,就是抗凝,抗氧化,防止纤维过度生长.个人认为,喝这个药的最好时机就是疤痕已经长稳固,而正在慢慢改建成与周围组织相似的过程中.而不再有变化的旧疤痕,效果不会很明显,细胞的改建活动已经不太活跃了. 7五灵脂丸:组成：五灵脂1500克。功效：活血破淤，软坚化滞。主治：瘢痕疙瘩。用法：研细末，炼蜜为丸，每丸3克。每次半丸至一丸半，日2次，温开水送下。 8茄子消瘢剂: 颜面或身体受伤青肿，可用老黄茄子极大者，切片如一指厚，新瓦焙研为未。欲卧时温酒调服7．5克。古人称应用此方，“一夜消尽且无痕迹”。出处：《胜金方》 9瘢痕疙瘩:香附9克，柴胡9克，川芎9克，熟地12克，当归12克，赤芍9克，穿山甲9克，夏枯草15克，梨树根45克。功效：调和营卫，舒畅气血。主治：瘢痕疙瘩，食道粘连，食道憩室。用法：将穿山甲、梨树根用清水浸泡30分钟，再入余药，煎煮30分钟，每剂煎2次，将2次煎液混合。每日1剂，早、晚各服1次，食道粘连者可日服3-5次 10防风、荆芥、丹参、白鲜皮;党参、淮山药、生地、熟地;桃仁、藏红花、浙贝母、昆布. 舅舅是皮肤科的老中医，因为一个同学长疤痕疙瘩，求治多方无效。想到了用中医方法祛疤，于是我找到舅舅，想了解一下中医方法怎么治。舅舅给我们分析得很详细。他说，中医强调治病必须审证求因，认为瘢痕疙瘩病变虽在体表，但根在体内，强调通过内服中药调理机体阴阳以治其本，中医的内治法主要有三：其一、内服防风、荆芥、丹参、白鲜皮等的“清热解毒、疏风散表、止痒法”；其二、内服党参、淮山药、生地、熟地等的“养阴易气、养血润燥法”；其三、内服桃仁、藏红花、浙贝母、昆布等的“活血化淤、软坚散结法”。中医治病不仅强调治本，也重视治标， 1用三七粉加醋治疗疤痕疙瘩的偏方后，抱着试度看的心情，先用家中的香醋加三七粉上了几天，没有什么感觉，又买了一并陈醋，将三七粉调成糊状，上药二周后，就见疤痕疙瘩上出现许多向针眼似的小孔，从里边流黄水，但上药时感到疼痛难忍，这时我想到上网查查，看到这个贴子说要用米醋加三七粉，于是又买了一并九度的白色米醋，续继上药，由于洗澡使疤痕疙瘩创面受到感染，化脓了，我又用盐水将脓洗去，将三七粉直接涂在创面上，数日结茄。末感染的疤痕休明显扁平，颜色也明显变浅了。我要告诉大家，要有信心，坚持上药一定会好的。