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1.
PLoS Genet ; 9(10): e1003773, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24146624

RESUMO

Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.


Assuntos
Metilação de DNA/genética , Epigênese Genética , Sequências de Repetição em Tandem/genética , Transcrição Gênica , Alelos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Mutação , Plantas Geneticamente Modificadas/genética , RNA/genética , RNA Interferente Pequeno/genética , Zea mays/genética , Zea mays/metabolismo
2.
PLoS Genet ; 8(10): e1002980, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23055943

RESUMO

To understand the molecular mechanisms underlying paramutation, we examined the role of Unstable factor for orange1 (Ufo1) in maintaining paramutation at the maize pericarp color1 (p1) and booster1 (b1) loci. Genetic tests revealed that the Ufo1-1 mutation disrupted silencing associated with paramutation at both p1 and b1. The level of up regulation achieved at b1 was lower than that at p1, suggesting differences in the role Ufo1-1 plays at these loci. We characterized the interaction of Ufo1-1 with two silenced p1 epialleles, P1-rr' and P1-pr(TP), that were derived from a common P1-rr ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; P1-rr' is paramutagenic to P1-rr, while P1-pr(TP) is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; P1-rr' exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while P1-pr(TP) was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the Ufo1-1 background; however, reactivation of P1-rr' was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated P1-pr(TP) was not affected. Interestingly, Ufo1-1-mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the Ufo1-1-reactivated p1 alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic p1 alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.


Assuntos
Inativação Gênica , Mutação , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/genética , Zea mays/metabolismo , Alelos , Metilação de DNA , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Ordem dos Genes , Histonas/metabolismo , Proteínas de Plantas/metabolismo , Locos de Características Quantitativas , Ativação Transcricional
3.
Genetics ; 226(1)2024 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-38169343

RESUMO

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr' state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr'. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr' epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr' sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.


Assuntos
Metilação de DNA , Zea mays , Zea mays/genética , Mutação , Plantas/genética , RNA , Elementos Facilitadores Genéticos , Alelos , Regulação da Expressão Gênica de Plantas
4.
Proc Natl Acad Sci U S A ; 107(12): 5516-21, 2010 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-20212123

RESUMO

Paramutation is the ability of specific DNA sequences to communicate in trans to establish meiotically heritable expression states. Paramutation at the maize b1 locus is mediated by seven unique noncoding transcribed tandem repeats of 853 bp that are required to establish and maintain the meiotically heritable expression and distinct chromatin states associated with b1 paramutation. In this study, we report the identification of a CXC-domain protein CBBP (CXC domain b1-repeat binding protein) that binds to a defined region within the b1 tandem repeat sequence in vivo and in vitro. When CBBP is expressed from a transgene in maize, it can induce a silent state at the b1 locus that is heritable in progeny no longer containing the transgene, and the silent epiallele is capable of silencing an active epiallele, characteristic of paramutation. Accumulation of the CBBP protein correlates with b1 silencing in transgenic and nontransgenic plants. The ability of CBBP to form multimers and to bind to the b1 tandem repeats suggests a model for counting the number of b1 repeats. In contrast to previously identified proteins involved in paramutation, CBBP does not share similarity to the known components of the Arabidopsis RNAi heterochromatin silencing pathway. Thus, this study defines another class of protein that is involved in heritable gene silencing.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Plantas/genética , Zea mays/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Inativação Gênica , Modelos Genéticos , Mutação , Fenótipo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequências de Repetição em Tandem , Técnicas do Sistema de Duplo-Híbrido , Zea mays/metabolismo
5.
Proc Natl Acad Sci U S A ; 107(29): 12986-91, 2010 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-20616013

RESUMO

Paramutation is the epigenetic transfer of information between alleles that leads to the heritable change of expression of one allele. Paramutation at the b1 locus in maize requires seven noncoding tandem repeat (b1TR) sequences located approximately 100 kb upstream of the transcription start site of b1, and mutations in several genes required for paramutation implicate an RNA-mediated mechanism. The mediator of paramutation (mop1) gene, which encodes a protein closely related to RNA-dependent RNA polymerases, is absolutely required for paramutation. Herein, we investigate the potential function of mop1 and the siRNAs that are produced from the b1TR sequences. Production of siRNAs from the b1TR sequences depends on a functional mop1 gene, but transcription of the repeats is not dependent on mop1. Further nuclear transcription assays suggest that the b1TR sequences are likely transcribed predominantly by RNA polymerase II. To address whether production of b1TR-siRNAs correlated with paramutation, we examined siRNA production in alleles that cannot undergo paramutation. Alleles that cannot participate in paramutation also produce b1TR-siRNAs, suggesting that b1TR-siRNAs are not sufficient for paramutation in the tissues analyzed. However, when b1TR-siRNAs are produced from a transgene expressing a hairpin RNA, b1 paramutation can be recapitulated. We hypothesize that either the b1TR-siRNAs or the dsRNA template mediates the trans-communication between the alleles that establishes paramutation. In addition, we uncovered a role for mop1 in the biogenesis of a subset of microRNAs (miRNAs) and show that it functions at the level of production of the primary miRNA transcripts.


Assuntos
Loci Gênicos/genética , Mutação/genética , RNA de Plantas/metabolismo , Zea mays/genética , Alelos , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , MicroRNAs/biossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA de Cadeia Dupla/biossíntese , RNA Interferente Pequeno/biossíntese , Sequências de Repetição em Tandem/genética , Transcrição Gênica , Transgenes/genética
6.
Nature ; 442(7100): 295-8, 2006 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-16855589

RESUMO

Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.


Assuntos
Mutagênese/genética , Mutação/genética , RNA Polimerase Dependente de RNA/metabolismo , Zea mays/enzimologia , Zea mays/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica de Plantas/genética , Dados de Sequência Molecular , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Sequências de Repetição em Tandem/genética , Transcrição Gênica/genética
7.
PLoS Genet ; 5(11): e1000725, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19936058

RESUMO

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Inativação Gênica , Genes Dominantes/genética , Mutação/genética , Subunidades Proteicas/genética , RNA Interferente Pequeno/metabolismo , Zea mays/enzimologia , Alelos , Motivos de Aminoácidos , Sequência de Aminoácidos , Pareamento de Bases , Sequência Conservada , RNA Polimerases Dirigidas por DNA/química , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Loci Gênicos/genética , Heterozigoto , Homozigoto , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/genética , Subunidades Proteicas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Sequências de Repetição em Tandem/genética , Transcrição Gênica , Transgenes/genética , Zea mays/genética , Zea mays/crescimento & desenvolvimento
8.
Proc Natl Acad Sci U S A ; 105(39): 14958-63, 2008 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-18815367

RESUMO

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-of-function mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic approximately 22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.


Assuntos
Proteínas de Plantas/fisiologia , RNA Interferente Pequeno/biossíntese , Zea mays/genética , Zea mays/metabolismo , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutação , Proteínas de Plantas/genética , RNA Polimerase Dependente de RNA/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos
9.
BMC Plant Biol ; 8: 33, 2008 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-18402703

RESUMO

BACKGROUND: Heterosis is the superior performance of F1 hybrid progeny relative to the parental phenotypes. Maize exhibits heterosis for a wide range of traits, however the magnitude of heterosis is highly variable depending on the choice of parents and the trait(s) measured. We have used expression profiling to determine whether the level, or types, of non-additive gene expression vary in maize hybrids with different levels of genetic diversity or heterosis. RESULTS: We observed that the distributions of better parent heterosis among a series of 25 maize hybrids generally do not exhibit significant correlations between different traits. Expression profiling analyses for six of these hybrids, chosen to represent diversity in genotypes and heterosis responses, revealed a correlation between genetic diversity and transcriptional variation. The majority of differentially expressed genes in each of the six different hybrids exhibited additive expression patterns, and approximately 25% exhibited statistically significant non-additive expression profiles. Among the non-additive profiles, approximately 80% exhibited hybrid expression levels between the parental levels, approximately 20% exhibited hybrid expression levels at the parental levels and ~1% exhibited hybrid levels outside the parental range. CONCLUSION: We have found that maize inbred genetic diversity is correlated with transcriptional variation. However, sampling of seedling tissues indicated that the frequencies of additive and non-additive expression patterns are very similar across a range of hybrid lines. These findings suggest that heterosis is probably not a consequence of higher levels of additive or non-additive expression, but may be related to transcriptional variation between parents. The lack of correlation between better parent heterosis levels for different traits suggests that transcriptional diversity at specific sets of genes may influence heterosis for different traits.


Assuntos
Perfilação da Expressão Gênica , Vigor Híbrido/genética , Zea mays/genética , Regulação da Expressão Gênica de Plantas , Hibridização Genética , Endogamia , Análise de Sequência com Séries de Oligonucleotídeos
10.
Genetics ; 162(2): 917-30, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12399399

RESUMO

Paramutation is an interaction between alleles that leads to a heritable change in the expression of one allele. In B'/B-I plants, B-I (high transcription) always changes to B' (low transcription). The new B' allele retains the low expression state in the next generation and paramutates B-I at a frequency of 100%. Comparisons of the structure and expression of B' with that of a closely related allele that does not participate in paramutation demonstrated that transcription from the same promoter-proximal sequences is not sufficient for paramutation. Fine-structure recombination mapping localized sequences required for B' expression and paramutation. The entire 110 kb upstream of the B' transcription start site was cloned and sequenced and the recombination breakpoints were determined for 12 recombinant alleles. Sequences required for expression and paramutation mapped to distinct regions, 8.5-49 kb and 93-106 kb upstream of the B' transcription start site, respectively. Sequencing and DNA blot analyses indicate that the B' region required for paramutation is mostly unique or low copy in the maize genome. These results represent the first example of long-distance regulatory elements in plants and demonstrate that paramutation is mediated by long-distance cis and trans interactions.


Assuntos
Genes Reguladores , Transcrição Gênica/genética , Zea mays/genética , Mapeamento Cromossômico , Mutação , Polimorfismo Genético , Recombinação Genética
11.
Cell Rep ; 9(1): 378-390, 2014 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-25284785

RESUMO

Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic analyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two subtypes of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Zea mays/metabolismo , Domínio Catalítico , Filogenia , Plantas Geneticamente Modificadas/metabolismo , Transcrição Gênica
12.
Genes (Basel) ; 4(2): 226-43, 2013 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-24705161

RESUMO

Short Interspersed Nuclear Elements (SINEs) are non-autonomous retrotransposons that comprise a large fraction of the human genome. SINEs are demethylated in human disease, but whether SINEs become transcriptionally induced and how the resulting transcripts may affect the expression of protein coding genes is unknown. Here, we show that downregulation of the mRNA of the tumor suppressor gene BRCA1 is associated with increased transcription of SINEs and production of sense and antisense SINE small RNAs. We find that BRCA1 mRNA is post-transcriptionally down-regulated in a Dicer and Drosha dependent manner and that expression of a SINE inverted repeat with sequence identity to a BRCA1 intron is sufficient for downregulation of BRCA1 mRNA. These observations suggest that transcriptional activation of SINEs could contribute to a novel mechanism of RNA mediated post-transcriptional silencing of human genes.

13.
Science ; 330(6004): 628-9, 2010 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-21030647

RESUMO

Paramutation refers to the process by which homologous DNA sequences communicate in trans to establish meiotically heritable expression states. Although mechanisms are unknown, current data are consistent with the hypothesis that the establishment and heritable transmission of specific chromatin states underlies paramutation. Transcribed, noncoding tandem repeats and proteins implicated in RNA-directed transcriptional silencing in plants and yeast are required for paramutation, yet the specific molecules mediating heritable silencing remain to be determined.


Assuntos
Cromatina/metabolismo , Epigênese Genética , Mutação , Interferência de RNA , RNA Interferente Pequeno/genética , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Divisão Celular , Cromatina/química , Cromatina/ultraestrutura , Metilação de DNA , DNA Fúngico/metabolismo , DNA de Plantas/metabolismo , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , RNA Fúngico/química , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequências de Repetição em Tandem , Zea mays/genética , Zea mays/metabolismo
14.
Cell ; 128(4): 641-5, 2007 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-17320501

RESUMO

Paramutation is the epigenetic transfer of information from one allele of a gene to another to establish a state of gene expression that is heritable for generations. RNA has recently emerged as a prominent mediator of this remarkable phenomenon in both maize and mice.


Assuntos
Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Hereditariedade , Mutação/genética , RNA/genética , Zea mays/genética , Alelos , Animais , Humanos , Camundongos
15.
Proc Natl Acad Sci U S A ; 99(9): 6130-5, 2002 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-11959901

RESUMO

Both paramutation and Mutator (Mu) transposon inactivation involve heritable changes in gene expression without concomitant changes in DNA sequence. The mechanisms by which these shifts in gene activity are achieved are unknown. Here we present evidence that these two phenomena are linked mechanistically. We show that mutation of a gene, modifier of paramutation 1 (mop1), which prevents paramutation at three different loci in maize, can reverse methylation of Mutator elements reliably. In mop1 mutant backgrounds, methylation of nonautonomous Mu elements can be reversed even in the absence of the regulatory MuDR element. Previously silenced MuDR elements are reactivated sporadically after multiple generations of exposure to mop1 mutations. MuDR methylation is separable from MuDR silencing, because removal of methylation does not cause immediate reactivation. The mop1 mutation does not alter the methylation of certain other transposable elements including those just upstream of a paramutable b1 gene. Our results suggest that the mop1 gene acts on a subset of epigenetically regulated sequences in the maize genome and paramutation and Mu element methylation require a common factor, which we hypothesize influences chromatin structure.


Assuntos
Elementos de DNA Transponíveis/genética , Mutação , Zea mays/genética , Cromatina/metabolismo , Cruzamentos Genéticos , DNA/metabolismo , Metilação de DNA , Inativação Gênica , Homozigoto
16.
Genes Dev ; 16(15): 1906-18, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12154122

RESUMO

Recombination mapping defined a 6-kb region, 100 kb upstream of the transcription start site, that is required for B-I enhancer activity and paramutation-a stable, heritable change in transcription caused by allele interactions in maize (Zea mays). In this region, B-I and B' (the only b1 alleles that participate in paramutation) have seven tandem repeats of an 853-bp sequence otherwise unique in the genome; other alleles have one. Examination of recombinant alleles with different numbers of tandem repeats indicates that the repeats are required for both paramutation and enhancer function. The 6-kb region is identical in B-I and B', showing that epigenetic mechanisms mediate the stable silencing associated with paramutation. This is the first endogenous gene for which sequences required for paramutation have been defined and examined for methylation and chromatin structure. The tandem repeat sequences are more methylated in B-I (high expressing) relative to B' (low expressing), opposite of the typical correlation. Furthermore, the change in repeat methylation follows establishment of the B' epigenetic state. B-I has a more open chromatin structure in the repeats relative to B'. The nuclease hypersensitivity differences developmentally precede transcription, suggesting that the repeat chromatin structure could be the heritable imprint distinguishing the two transcription states.


Assuntos
Cromatina/genética , DNA de Plantas/genética , Proteínas de Plantas/genética , Sequências de Repetição em Tandem , Fatores de Transcrição/genética , Transcrição Gênica/genética , Zea mays/genética , Alelos , Sequência de Bases , Mapeamento Cromossômico , Sequência Consenso , Metilação de DNA , Elementos Facilitadores Genéticos , Flavonoides/biossíntese , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Pigmentação/genética , Recombinação Genética , Homologia de Sequência do Ácido Nucleico
17.
Plant Cell ; 16(2): 450-64, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14742877

RESUMO

The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.


Assuntos
Antocianinas/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Plantas/genética , Sementes/genética , Zea mays/genética , Alelos , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Extensões da Superfície Celular/genética , Extensões da Superfície Celular/fisiologia , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Sequências Repetitivas de Aminoácidos/fisiologia , Sementes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/metabolismo
18.
Plant Physiol ; 128(3): 896-910, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11891246

RESUMO

Assembly of 73,000 expressed sequence tags (ESTs) representing multiple organs and developmental stages of maize (Zea mays) identified approximately 22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4% to 20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of micro-array slides were fabricated, one containing 5,534 ESTs from 10- to 14-d-old endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear poly(A(+)) RNA. Although the 10- to 14-d-old postpollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low-abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross hybridization among gene family members can overestimate the overlap in genes expressed in micro-array hybridization experiments.


Assuntos
Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA de Plantas/genética , Zea mays/genética , Mapeamento de Sequências Contíguas , Genoma de Planta , Família Multigênica
19.
Plant Physiol ; 132(2): 907-25, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12805620

RESUMO

Histone proteins play a central role in chromatin packaging, and modification of histones is associated with chromatin accessibility. SET domain [Su(var)3-9, Enhancer-of-zeste, Trithorax] proteins are one class of proteins that have been implicated in regulating gene expression through histone methylation. The relationships of 22 SET domain proteins from maize (Zea mays) and 32 SET domain proteins from Arabidopsis were evaluated by phylogenetic analysis and domain organization. Our analysis reveals five classes of SET domain proteins in plants that can be further divided into 19 orthology groups. In some cases, such as the Enhancer of zeste-like and trithorax-like proteins, plants and animals contain homologous proteins with a similar organization of domains outside of the SET domain. However, a majority of plant SET domain proteins do not have an animal homolog with similar domain organization, suggesting that plants have unique mechanisms to establish and maintain chromatin states. Although the domains present in plant and animal SET domain proteins often differ, the domains found in the plant proteins have been generally implicated in protein-protein interactions, indicating that most SET domain proteins operate in complexes. Combined analysis of the maize and Arabidopsis SET domain proteins reveals that duplication of SET domain proteins in plants is extensive and has occurred via multiple mechanisms that preceded the divergence of monocots and dicots.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Duplicação Gênica , Regulação da Expressão Gênica de Plantas/fisiologia , Histona-Lisina N-Metiltransferase , Metiltransferases/genética , Proteínas de Plantas/genética , Transcrição Gênica , Zea mays/genética , Sequência de Aminoácidos , Animais , Arabidopsis/classificação , Proteínas de Arabidopsis/química , Sequência de Bases , Análise por Conglomerados , Primers do DNA , Histona Metiltransferases , Metiltransferases/química , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas Metiltransferases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Zea mays/classificação
20.
J Biol Chem ; 279(46): 48205-13, 2004 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-15347654

RESUMO

The R2R3 MYB transcription factor C1 requires the basic helix-loop-helix factor R as an essential co-activator for the transcription of maize anthocyanin genes. In contrast, the R2R3 MYB protein P1 activates a subset of the C1-regulated genes independently of R. Substitution of six amino acids in P1 with the C1 amino acids results in P1(*), whose activity on C1-regulated and P1-regulated genes is R-dependent or R-enhanced, respectively. We have used P1(*) in combination with various promoters to uncover two mechanisms for R function. On synthetic promoters that contain only C1/P1 binding sites, R is an essential co-activator of C1. This function of R is unlikely to simply be the result of an increase in the C1 DNA-binding affinity, since transcriptional activity of a C1 mutant that binds DNA at a higher affinity, comparable with P1, remains R-dependent. The differential transcriptional activity of C1 fusions with the yeast Gal4 DNA-binding domain in yeast and maize cells suggests that part of the function of R is to relieve C1 from a plant-specific inhibitor. A second function of R requires cis-regulatory elements in addition to the C1/P1 DNA-binding sites for R-enhanced transcription of a1. We hypothesize that R functions in this mode by binding or recruiting additional factors to the anthocyanin regulatory element conserved in the promoters of several anthocyanin genes. Together, these findings suggest a model in which combinatorial interactions with co-activators enable R2R3 MYB factors with very similar DNA binding preferences to discriminate between target genes in vivo.


Assuntos
Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Sequências Hélice-Alça-Hélice , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flavonoides/biossíntese , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido
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