5-halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2)

Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals.     

On how mammalian transcription factors recognize methylated DNA

DNA methylation is an epigenetic mark that is essential for the development of mammals; it is frequently altered in diseases ranging from cancer to psychiatric disorders. The presence of DNA methylation attracts specialized methyl-DNA binding factors that can then recruit chromatin modifiers. These methyl-CpG binding proteins (MBPs) have key biological roles and can be classified into three structural families: methyl-CpG binding domain (MBD), zinc finger, and SET and RING finger-associated (SRA) domain. The structures of MBD and SRA proteins bound to methylated DNA have been previously determined and shown to exhibit two very different modes of methylated DNA recognition. The last piece of the puzzle has been recently revealed by the structural resolution of two different zinc finger proteins, Kaiso and ZFP57, in complex with methylated DNA. These structures show that the two methyl-CpG binding zinc finger proteins adopt differential methyl-CpG binding modes. Nonetheless, there are similarities with the MBD proteins suggesting some commonalities in methyl-CpG recognition across the various MBP domains. These fresh insights have consequences for the analysis of the many other zinc finger proteins present in the genome, and for the biology of methyl-CpG binding zinc finger proteins.     

Treatment of tumor cells with histone deacetylase inhibitors results in altered recruitment of methyl-CpG binding proteins to a methylated CpG island

When human cancer cells with silencing of the CDH1 gene associated with CpG island methylation and histone deacetylation were treated with histone deacetylase inhibitors, alteration in recruitment of methyl-CpG binding proteins (MBPs) to the methylated CDH1-CpG island was observed, as well as altered histone acetylation status. This change was independent of the histone deacetylase inhibitor used. These results suggest that histone hyperacetylation provides a more open chromatin structure conformation for the recruitment of additional MBPs.     

In Vitro Synthesis of the Four Mouse Myelin Basic Proteins: Evidence for the Lack of a Metabolic Relationship

Abstract: Studies on the synthesis of the four immunologically related mouse myelin basic proteins (MBPs) were carried out to determine if these proteins were metabolically related. Two in vitro systems were used: (a) a homologous brain system consisting of free polysomes, pH 5 enzymes, and initiation factors; and (b) a reticulocyte lysate system directed with mRNA and supplemented with brain factors. Incorporation of [³⁵S]methionine into the four MBPs (14K, 17K, 18.5K, and 21.5K) was detected by immunoprecipitation of the in vitro products of synthesis followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The four MBPs were identified by cross-reactivity with purified anti-MBP antibodies and their apparent molecular weights in SDS gels. Synthesis of all four proteins was detected in both systems soon after the incubations were begun. The kinetics of the labeling of the proteins showed no evidence of a precursor-product relationship (i.e., 21.5K→ 18.5K; 17K → 14K) in either system. Inhibition studies with puromycin and "chase" experiments with unlabeled methionine demonstrated that neither system contained posttranslational "processing" activity. Thus, the 21.5K and 17K proteins were not being processed into the 18.5K and 14K MBPs by either . in vitro system. Detection of the synthesis of all four proteins in the reticulocyte system programmed with brain mRNA indicates that the four proteins are probably coded for by separate mRNAs. This conclusion was supported by studies using polyribosomes separated into different size classes, which suggest that the mRNAs for the four proteins may be translated on proteins of differing size class. It is proposed, therefore, that the four MBPs are the primary translation products of independent brain mRNAs and are not metabolically related.     

In Vitro Synthesis of the Myelin Basic Proteins: Subcellular Site of Synthesis

Abstract: Brains of 3-week-old C57BL/6J mice were homogenized and fractionated into several subcellular components, each of which was examined for ability to synthesize the myelin basic proteins (MBPs) in vitro. Myelin basic proteins were purified from incubation mixtures by conventional means. That the products of synthesis were the myelin basic proteins was established by solubility at pH 3, co-chromatography with authentic proteins on carboxymethylcellulose and co-migration with standards in two different polyacrylamide gel electrophoretic systems. The fractions examined for their ability to synthesize MBPs were the whole homogenate, postnuclear supernatant, postmitochondrial supernatant, crude mitochondrial pellet, free ribosomes and bound ribosomes. Although there was no requirement for exogenous energy sources for protein synthesis in the whole homogenate, as the homogenate was fractionated an increasing requirement emerged. Most of the label in the MBP preparations from whole homogenate and postnuclear supernatant incubations migrated with the large (L) and small (S) MBPs on gel electrophoresis; however, as the homogenate was subfractionated and incubated, a greater percentage of the label migrated more slowly than L and S on acetic acid-urea gels. To show synthesis of the MBPs the L and S bands were cut out of these gels and rerun on sodium dodecylsulfate gels. Alternatively, MBP preparations were subjected directly to two-dimensional gel electrophoresis and the bands corresponding to L and S were excised and counted. With this method only the whole homogenate, postnuclear supernatant, postmitochondrial supernatant and free ribosomes were observed to synthesize the MBPs in vitro. The "bound" ribosomes were not observed to synthesize significant amounts of the MBPs, incubated either intact or released from the membrane. It was concluded that the free ribosomes are the principal site of synthesis of the myelin basic proteins in the brain.     

RNA-directed DNA methylation mediated by DRD1 and Pol IVb: a versatile pathway for transcriptional gene silencing in plants

RNA-directed DNA methylation, which is one of several RNAi-mediated pathways in the nucleus, has been highly elaborated in the plant kingdom. RNA-directed DNA methylation requires for the most part conventional DNA methyltransferases, histone modifying enzymes and RNAi proteins; however, several novel, plant-specific proteins that are essential for this process have been identified recently. DRD1 (defective in RNA-directed DNA methylation) is a putative SWI2/SNF2-like chromatin remodelling protein; DRD2 and DRD3 (renamed NRPD2a and NRPD1b, respectively) are subunits of Pol IVb, a putative RNA polymerase found only in plants. Interestingly, DRD1 and Pol IVb appear to be required not only for RNA-directed de novo methylation, but also for full erasure of methylation when the RNA trigger is withdrawn. These proteins thus have the potential to facilitate dynamic regulation of DNA methylation. Prominent targets of RNA-directed DNA methylation in the Arabidopsis thaliana genome include retrotransposon long terminal repeats (LTRs), which have bidirectional promoter/enhancer activities, and other types of intergenic transposons and repeats. Intergenic solitary LTRs that are targeted for reversible methylation by the DRD1/Pol IVb pathway can potentially act as switches or rheostats for neighboring plant genes. The resulting alterations in gene expression patterns may promote physiological flexibility and adaptation to the environment.     

In vivo phosphorylation of myelin basic proteins: age-related differences in 32P incorporation

Myelin basic proteins (MBPs) are phosphoproteins of central and peripheral nervous system myelin. We studied the phosphorylation of mouse MBPs in vivo at three different stages of development (12, 30, and 50 days) and found age-related differences in the incorporation of 32P into MBPs. At all ages studied, significant amounts of 32P were found in the MBPs as early as 1 min after intracranial injection of isotope. Incorporation of radioactive phosphate into MBPs proceeded rapidly and the resultant specific radioactivity (SA) of 32P-labeled MBPs appeared to be related to the SA of the acid-soluble phosphate pool of myelin. Changes in the SA of the myelin acid-soluble phosphate pool were observed in a 30 min time course of labeling in vivo in 50-day mice. Coincident changes were observed in the SA of the MBPs. Similar but less pronounced changes were seen in the SA of the polyphosphoinositides (PPIs) indicating that the turnover of the PPI phosphate groups is slower than the MBP phosphates or that the PPI phosphates are drawn from additional or different pools than the MBP phosphates. The phosphorylation of MBPs in developmentally related myelin fractions is investigated in a comparison paper (J. B. Ulmer and P. E. Braun (1986) Dev. Biol. 117, 502-510).     

Synthesis and incorporation of myelin polypeptides into CNS myelin

The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath.     

Viral genome methylation as an epigenetic defense against geminiviruses

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.     

Arginine methylation of RNA-binding proteins regulates cell function and differentiation

Arginine methylation is a post-translational modification that regulates protein function. RNA-binding proteins are an important class of cell-function mediators, some of which are methylated on arginine. Early studies of RNA-binding proteins and arginine methylation are briefly introduced, and the enzymes that mediate this post-translational modification are described. We review the most common RNA-binding domains and briefly discuss how they associate with RNAs. We address the following groups of RNA-binding proteins: hnRNP, Sm, Piwi, Vasa, FMRP, and HuD. hnRNPs were the first RNA-binding proteins found to be methylated on arginine. The Sm proteins function in RNA processing and germ cell specification. The Piwi proteins are largely germ cell specific and are also required for germ cell production, as is Vasa. FMRP participates in germ cell formation in Drosophila, but is more widely known for its neuronal function. Similarly, HuD plays a role in nervous system development and function. We review the effects of arginine methylation on the function of each protein, then conclude by addressing remaining questions and future directions of arginine methylation as an important and emerging area of regulation.     

Expression of Myelin Proteolipid Protein and Basic Protein in Normal and Dysmyelinating Mutant Mice

Expression of myelin proteins was studied in the brains of 21-day-old normal mice and three dysmyelinating mutants-jimpy, quaking, and shiverer. Total brain polyribosomes and poly(A)+ mRNA were translated in two cell-free systems and the levels of synthesis of the myelin basic proteins (MBPs) and proteolipid protein (PLP) were determined. Synthesis of the MBPs in quaking homozygotes was at or above normal levels but PLP synthesis was significantly reduced to approximately 15% of control values, indicating independent effects on the expression of these proteins in this mutant. Immunoblot analysis of 21-day-old quaking brain homogenates showed a reduction in the steady-state levels of MBPs and PLP, suggesting a failure of newly synthesized MBPs to be incorporated into a stable membrane structure such as myelin. In the shiverer mutant very little synthesis of MBPs was observed, whereas greater synthesis of PLP occurred (approximately 50% of control). Almost no MBP, and low levels of PLP, were detected in the immunoblots, suggesting the possibility of a partial failure of PLP to be assembled into myelin in shiverer. In the jimpy mutant, low levels of MBP synthesis were observed in vitro (approximately 26% of controls) and very little synthesis of PLP was evident. The immunoblots of 21-day jimpy brain homogenates revealed no appreciable steady-state levels of PLP or MBP, again indicating that most newly synthesized MBPs were not incorporated into a stable membrane structure in this mutant. In sum, the data show that in the three cases examined, the mutation appears to affect the expression of the MBPs and PLP independently. Furthermore, regardless of their absolute levels of synthesis these proteins may or may not be assembled into myelin.