Methylation of the Protein Phosphatase 2A Catalytic Subunit Is Essential for Association of Bα Regulatory Subunit But Not SG2NA, Striatin, or Polyomavirus Middle Tumor Antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Bα subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Bα subunit binding without greatly affecting methylation, indicating that Bα subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Bα subunit but not the amount that could be coimmunoprecipitated with Aα subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Bα subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Bα subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.

Methylation by a mutant T2 DNA [N6-adenine] methyltransferase expands the usage of RecA-assisted endonuclease (RARE) cleavage

Properties of a mutant bacteriophage T2 DNA [N6-adenine] methyltransferase (T2 Dam MTase) have been investigated for its potential utilization in RecA-assisted restriction endonuclease (RARE) cleavage. Steady-state kinetic analyses with oligonucleotide duplexes revealed that, compared to wild-type T4 Dam, both wild-type T2 Dam and mutant T2 Dam P126S had a 1.5-fold higher kcat in methylating canonical GATC sites. Additionally, T2 Dam P126S showed increased efficiencies in methylation of non-canonical GAY sites relative to the wild-type enzymes. In agreement with these steady-state kinetic data, when bacteriophage λ DNA was used as a substrate, maximal protection from restriction nuclease cleavage in vitro was achieved on the sequences GATC, GATN and GACY, while protection of GACR sequences was less efficient. Collectively, our data suggest that T2 Dam P126S can modify 28 recognition sequences. The feasibility of using the mutant enzyme in RARE cleavage with BclI and EcoRV endonucleases has been shown on phage λ DNA and with BclI and DpnII endonucleases on yeast chromosomal DNA embedded in agarose.

Methylation of coding region alone inhibits gene expression in plant protoplasts.

Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation.

Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development.

Arabidopsis plants transformed with an antisense construct of an Arabidopsis methyltransferase cDNA (METI) have reduced cytosine methylation in CG dinucleotides. Methylation levels in progeny of five independent transformants ranged from 10% to 100% of the wild type. Removal of the antisense construct by segregation in sexual crosses did not fully restore methylation patterns in the progeny, indicating that methylation patterns are subject to meiotic inheritance in Arabidopsis. Plants with decreased methylation displayed a number of phenotypic and developmental abnormalities, including reduced apical dominance, smaller plant size, altered leaf size and shape, decreased fertility, and altered flowering time. Floral organs showed homeotic transformations that were associated with ectopic expression of the floral homeotic genes AGAMOUS and APETALA3 in leaf tissue. These observations suggest that DNA methylation plays an important role in regulating many developmental pathways in plants and that the developmental abnormalities seen in the methyltransferase antisense plants may be due to dysregulation of gene expression.

Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.

5-Methyl-2'-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.

Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments.

Insertion of foreign DNA into an established mammalian genome can extensively alter the patterns of cellular DNA methylation. Adenovirus type 12 (Ad12)-transformed hamster cells, Ad12-induced hamster tumor cells, or hamster cells carrying integrated DNA of bacteriophage lambda were used as model systems. DNA methylation levels were examined by cleaving cellular DNA with Hpa II, Msp I, or Hha I, followed by Southern blot hybridization with 32P-labeled, randomly selected cellular DNA probes. For several, but not all, cellular DNA segments investigated, extensive increases in DNA methylation were found in comparison with the methylation patterns in BHK21 or primary Syrian hamster cells. In eight different Ad12-induced hamster tumors, moderate increases in DNA methylation were seen. Increased methylation of cellular genes was also documented in two hamster cell lines with integrated Ad12 DNA without the Ad12-transformed phenotype, in one cloned BHK21 cell line with integrated plasmid DNA, and in at least three cloned BHK21 cell lines with integrated lambda DNA. By fluorescent in situ hybridization, the cellular hybridization probes were located to different hamster chromosomes. The endogenous intracisternal A particle genomes showed a striking distribution on many hamster chromosomes, frequently on their short arms. When BHK21 hamster cells were abortively infected with Ad12, increases in cellular DNA methylation were not seen. Thus, Ad12 early gene products were not directly involved in increasing cellular DNA methylation. We attribute the alterations in cellular DNA methylation, at least in part, to the insertion of foreign DNA. Can alterations in the methylation profiles of hamster cellular DNA contribute to the generation of the oncogenic phenotype?

Hairpins are formed by the single DNA strands of the fragile X triplet repeats: structure and biological implications.

Inordinate expansion and hypermethylation of the fragile X DNA triplet repeat, (GGC)n.(GCC)n, are correlated with the ability of the individual G- and C-rich single strands to form hairpin structures. Two-dimensional NMR and gel electrophoresis studies show that both the G- and C-rich single strands form hairpins under physiological conditions. This propensity of hairpin formation is more pronounced for the C-rich strand than for the G-rich strand. This observation suggests that the C-rich strand is more likely to form hairpin or "slippage" structure and show asymmetric strand expansion during replication. NMR data also show that the hairpins formed by the C-rich strands fold in such a way that the cytosine at the CpG step of the stem is C.C paired. The presence of a C.C mismatch at the CpG site generates local flexibility, thereby providing analogs of the transition to the methyltransferase. In other words, the hairpins of the C-rich strand act as better substrates for the human methyltransferase than the Watson-Crick duplex or the G-rich strand. Therefore, hairpin formation could account for the specific methylation of the CpG island in the fragile X repeat that occurs during inactivation of the FMR1 gene during the onset of the disease.

Preferential isolation of DNA fragments associated with CpG islands.

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.

Xce haplotypes show modified methylation in a region of the active X chromosome lying 3' to Xist.

During early mammalian embryogenesis, one of the two X chromosomes in somatic cells of the female becomes inactivated through a process that is thought to depend on a unique initiator region, the X-chromosome inactivation center (Xic). The recently characterized Xist sequence (X-inactive-specific transcript) is thought to be a possible candidate for Xic. In mice a further genetic element, the X chromosome-controlling element (Xce), is also known to influence the choice of which of the two X chromosomes is inactivated. We report that a region of the mouse X chromosome lying 15 kb distal to Xist contains several sites that show hypermethylation specifically associated with the active X chromosome. Analysis of this region in various Xce strains has revealed a correlation between the strength of the Xce allele carried and the methylation status of this region. We propose that such a region could be involved in the initial stages of the inactivation process and in particular in the choice of which of the two X chromosomes present in a female cell will be inactivated.

Investigação de mecanismos genéticos e epigenéticos em distúrbios do crescimento humano

Parcela considerável de pacientes com distúrbios de crescimento não têm a causa de seus quadros clínicos estabelecida, incluindo aproximadamente 50% dos pacientes com diagnóstico clínico de síndrome de Silver−Russell (SRS) e 10-20% dos pacientes com síndrome de Beckwith-Wiedemann (BWS). O objetivo deste estudo foi investigar as causas genéticas e epigenéticas de distúrbios de crescimento, de etiologia desconhecida, numa contribuição para o entendimento de mecanismos que regulam o crescimento. O estudo compreendeu: (1) a investigação de microdesequilíbrios cromossômicos, por aCGH; (2) a análise do perfil de expressão alelo-específica de genes sujeitos a imprinting (IG), por pirossequenciamento (PSQ) ou sequenciamento de Sanger; (3) a investigação do padrão de metilação global em pacientes com restrição de crescimento, utilizando microarray de metilação. A casuística constituiu-se de 41 pacientes não aparentados, com distúrbios de crescimento, de etiologia desconhecida: (1) 25, com hipótese diagnóstica de SRS; (2) seis, com restrição de crescimento intrauterino e peso ao nascimento abaixo do 10º percentil, associados a outros sinais clínicos; (3) sete, com hipótese diagnóstica de BWS; e (4) três, com macrossomia pré-natal ou pós-natal, associada a outros sinais. A investigação de microdesequilíbrios cromossômicos foi realizada em 40 pacientes. Foram detectadas 58 variantes raras em 30/40 pacientes (75%): 40 foram consideradas provavelmente benignas (18 pacientes, 45%), 12, com efeito patogênico desconhecido (11 pacientes, 27,5%), duas, provavelmente patogênicas (um paciente, 2,5%) e quatro, patogênicas (três pacientes, 7,5%). Essas frequências são comparáveis àquelas descritas em estudos que investigaram CNV em grupos de pacientes com distúrbios de crescimento e outras alterações congênitas, incluindo SRS, e mostram a importância da investigação de microdesequilíbrios cromossômicos nesses pacientes. A diversidade dos microdesequilíbrios cromossômicos identificados é reflexo da heterogeneidade clínica das casuísticas. Neste estudo, muitos dos pacientes com hipótese diagnóstica de SRS e BWS apresentavam sinais clínicos atípicos, explicando a ausência neles das alterações (epi)genéticas que causam essas síndromes. A identificação de CNV características de outras síndromes reflete a sobreposição de sinais clínicos com BWS e SRS. A análise do perfil de expressão alelo-específica de IG foi realizada em um subgrupo de 18 pacientes com restrição de crescimento. Trinta IG com função em proliferação celular, crescimento fetal ou neurodesenvolvimento foram inicialmente selecionados. Após seleção de SNP transcritos com alta frequência na população, genotipagem de pacientes, genitores e indivíduos controle, determinação da expressão dos IG em sangue periférico e seu padrão de expressão (mono ou bialélico), 13 IG, expressos no sangue, tiveram a expressão alelo-específica avaliada, sete deles por PSQ e seis por sequenciamento de Sanger. Alterações no perfil de expressão de dois genes, de expressão normalmente paterna, foram detectadas em 4/18 pacientes (22%). Este estudo é o primeiro a utilizar pirossequenciamento e sequenciamento de Sanger na avaliação do perfil de expressão alelo-específica de IG, em pacientes com restrição de crescimento. Apesar de terem limitações, ambas as técnicas mostraram-se robustas e revelaram alterações de expressão alélica interessantes; entretanto, a relação dessas alterações com o quadro clínico dos pacientes permanece por esclarecer. A investigação da metilação global do DNA foi realizada em subgrupo de 21 pacientes com restrição de crescimento e em 24 indivíduos controle. Dois tipos de análise foram realizados: (1) análise diferencial de grupo e (2) análise diferencial individual. Na primeira análise, em que foi comparado o padrão de metilação do grupo de pacientes com quadro clínico sugestivo de SRS (n=16) com o do grupo controle (n=24), não houve indicação de hipo ou hipermetilação global no grupo SRS. Na segunda análise, foi comparado o padrão de metilação de cada um dos 21 pacientes com restrição de crescimento e dos 24 indivíduos controle, com o padrão de metilação do grupo controle. O número médio de CpG hipermetilados e de segmentos diferencialmente metilados (SDM) foi significativamente maior nos pacientes. Foram identificados 82 SDM hipermetilados, estando 57 associados a gene(s) (69,5%), em 16 pacientes, e 51 SDM hipometilados, 41 deles associados a gene(s) (80,4%), em 10 pacientes. A análise de ontologia genética dos 61 genes associados aos SDM hipo ou hipermetilados nos pacientes destacou genes que atuam no desenvolvimento e na morfogênese do sistema esquelético e de órgãos fetais, e na regulação da transcrição gênica e de processos metabólicos. Alterações de metilação em genes que atuam em processos de proliferação e diferenciação celulares e crescimento foram identificadas em 9/20 dos pacientes (45%), sugerindo implicação clínica. Não foi detectada alteração epigenética comum aos pacientes com diagnóstico clínico de SRS, explicável provavelmente pela heterogeneidade clínica. A investigação de metilação global, utilizando microarray, produziu novos dados que podem contribuir para a compreensão de mecanismos moleculares que influenciam o crescimento pré- e pós-natal. Na translocação aparentemente equilibrada - t(5;6)(q35.2;p22.3)dn, detectada em paciente com suspeita clínica de SRS, a interrupção de um gene, pela quebra no cromossomo 6, pode ser a causa do quadro clínico; alternativamente, a translocação pode ter impactado a regulação de genes de desenvolvimento localizados próximos aos pontos de quebra. A análise de expressão em sangue periférico mostrou que os níveis de cDNA do gene, interrompido pelo ponto de quebra da translocação, estavam reduzidos à metade. Além de sinais típicos da SRS, a paciente apresentava algumas características clínicas sugestivas de displasia cleidocraniana. Assim, a translocação t(5;6) pode ter alterado a interação de genes de desenvolvimento e seus elementos reguladores, levando à desregulação de sua expressão espaço-temporal, e resultando num fenótipo atípico, com características sobrepostas de mais de uma síndrome genética

Influência da deficiência ou suplementação com selênio durante o período gestacional de ratas na suscetibilidade da progênie feminina à carcinogênese mamária

Fatores dietéticos como o selênio (Se) são apontados como importantes moduladores do risco de desenvolvimento do câncer de mama. Essa neoplasia pode apresentar sua origem no início do desenvolvimento e, assim, a alimentação materna poderia ter importantes repercussões na programação fetal da doença. A fim de verificar se diferentes concentração de selênio na dieta materna poderiam programar o risco da progênie feminina ao câncer de mama, ratas foram alimentadas com ração contendo 0,15 (CO), 1,0 (SUP) ou 0,05 (DEF) ppm de Se durante a gestação e sua progênie feminina iniciada com DMBA. A progênie do grupo SUP apresentou menor suscetibilidade à carcinogênese, indicado pelo menor número médio e multiplicidade de adenocarcinomas mamários (p< 0,05), enquanto a do grupo DEF apresentou maior suscetibilidade à carcinogênese, indicado pela maior incidência dos mesmos (p< 0,05). Mães do grupo DEF apresentaram menor concentração de Se no sangue (p< 0,05) e sua prole apresentou menor atividade da enzima GPx1 (p< 0,05). Além disso, observou-se na glândula mamária da progênie de 50 dias menor expressão (western blot e qPCR) de ERα, Her-2, EGFR e Ras no grupo SUP em comparação aos grupos CO e DEF (p< 0,05). Analisou-se, ainda, o padrão de metilação global do DNA (HPLC-DAD), expressão das enzimas DNMT1, 3a e 3b (qPCR), o padrão global de modificações pós traducionais em histonas (western blot) e o padrão de metilação da região promotora do gene Erα (modificação com bissulfito e pirossequenciamento) na glândula mamária da progênie de 50 dias. Não houve diferença no padrão de metilação global do DNA e expressão das enzimas DNMTs (p>0,05). Houve aumento na expressão de H4K16 acetilada nos grupos SUP e DEF (p< 0,05). Finalmente, em comparação a progênie do grupo DEF, a do grupo SUP apresentou região promotora de Erα com aumento marginal (p=0,07) na metilação de dois dinucleotídeos CpG. Conclui-se que o consumo de diferentes concentrações de Se na dieta materna tem impacto sobre a suscetibilidade da progênie ao câncer de mama na vida adulta através da modulação da expressão de receptores e oncogenes relacionados ao desenvolvimeto dessa neoplasia, além da influência em processos epigenéticos. Tais resultados apontam para a existência de uma \"janela de programação\" no início do desenvolvimento sensível a ação do Se, resultando em diminuição do risco de câncer de mama quando suplementado na dieta materna e o inverso quando de sua deficiencia.

Key factors involved in stress-induced microspore embryogenesis in barley and rapeseed: DNA methylation, arabinogalactan proteins and auxin

La embriogénesis de microsporas es un proceso in vitro en el que la microspora o polen inmaduro, mediante la aplicación de un tratamiento de estrés se reprograma y abandona su ruta de desarrollo gametofítico para iniciar la ruta embriogénica, dando lugar a embriones y plantas haploides y doble-haploides. Este proceso es de gran interés básico y aplicado en biotecnología y mejora vegetal para la obtención rápida de nuevas variedades, sin embargo aún tiene importantes limitaciones en su explotación por su baja eficiencia en muchas especies de interés económico. La limitación en la aplicación de este proceso es debida a que los mecanismos de inducción y progresión de la embriogénesis de microsporas no están todavía completamente dilucidados. La monocotiledónea Hordeum vulgare (cebada) y la dicotiledónea Brassica napus (colza) son especies modelo para este proceso, en las cuales se induce embriogénesis directa en cultivos de microsporas aisladas en medio líquido, mediante tratamientos de estrés con diferentes temperaturas...