Recognition of methylated DNA through methyl-CpG binding domain proteins

DNA methylation is a key regulatory control route in epigenetics, involving gene silencing and chromosome inactivation. It has been recognized that methyl-CpG binding domain (MBD) proteins play an important role in interpreting the genetic information encoded by methylated DNA (mDNA). Although the function of MBD proteins has attracted considerable attention and is well characterized, the mechanism underlying mDNA recognition by MBD proteins is still poorly understood. In this article, we demonstrate that the methyl-CpG dinucleotides are recognized at the MBD-mDNA interface by two MBD arginines through an interplay of hydrogen bonding and cation-π interaction. Through molecular dynamics and quantum-chemistry calculations we investigate the methyl-cytosine recognition process and demonstrate that methylation enhances MBD-mDNA binding by increasing the hydrophobic interfacial area and by strengthening the interaction between mDNA and MBD proteins. Free-energy perturbation calculations also show that methylation yields favorable contribution to the binding free energy for MBD-mDNA complex.     

Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.     

Towards an understanding of DNA recognition by the methyl-CpG binding domain 1

CpG methylation determines a variety of biological functions of DNA. The methylation signal is interpreted by proteins containing a methyl-CpG binding domain (MBDs). Based on the NMR structure of MBD1 complexed with methylated DNA we analysed the recognition mode by means of molecular dynamics simulations. As the protein is monomeric and recognizes a symmetrically methylated CpG step, the recognition mode is an asymmetric one. We find that the two methyl groups do not contribute equally to the binding energy. One methyl group is associated with the major part of the binding energy and the other one nearly does not contribute at all. The contribution of the two cytosine methyl groups to binding energy is calculated to be -3.6 kcal/mol. This implies a contribution of greater than two orders of magnitude to the binding constant. The conserved amino acid Asp32 is known to be essential for DNA binding by MBD1, but so far no direct contact with DNA has been observed. We detected a direct DNA base contact to Asp32. This could be the main reason for the importance of this amino acid. MBD contacts DNA exclusively in the major groove, the minor groove is reserved for histone contacts. We found a deformation of the minor groove shape due to complexation by MBD1, which indicates an information transfer between the major and the minor groove.     

The methyl-CpG binding domain and the evolving role of DNA methylation in animals

DNA methylation occurs in bacteria, fungi, plants and animals, however its role varies widely among different organisms. Even within animal genomes, methylation patterns vary substantially from undetectable in nematodes, to global methylation in vertebrate genomes. The number and variety of proteins containing methyl-CpG binding domains (MBDs) that are encoded in animal genomes also varies, with a general correlation between the extent of genomic methylation and the number of MBD proteins. We describe here the evolution of the MBD proteins and argue that the vertebrate MBD complement evolved to exploit the benefits and protect against the dangers of a globally methylated genome.     

PRR5 regulates phosphorylation,nuclear import and subnuclear localization of TOC1 in the Arabidopsis circadian clock

Many core oscillator components of the circadian clock are nuclear localized but how the phase and rate of their entry contribute to clock function is unknown. TOC1/PRR1, a pseudoresponse regulator (PRR) protein, is a central element in one of the feedback loops of the Arabidopsis clock, but how it functions is unknown. Both TOC1 and a closely related protein, PRR5, are nuclear localized, expressed in the same phase, and shorten period when deficient, but their molecular relationship is unclear. Here, we find that both proteins interact in vitro and in vivo through their conserved N‐termini. TOC1–PRR5 oligomerization enhances TOC1 nuclear accumulation two‐fold, most likely through enhanced nuclear import. In addition, PRR5 recruits TOC1 to large subnuclear foci and promotes phosphorylation of the TOC1 N‐terminus. Our results show that nuclear TOC1 is essential for normal clock function and reveal a mechanism to enhance phase‐specific TOC1 nuclear accumulation. Interestingly, this process of regulated nuclear import is reminiscent of similar oligomeric pairings in animal clock systems (e.g. timeless/period and clock/cycle), suggesting evolutionary convergence of a conserved mechanism across kingdoms.     

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.