Missense Variants Reveal Functional Insights Into the Human ARID Family of Gene Regulators.

Missense variants are alterations to protein coding sequences that result in amino acid substitutions. They can be deleterious if the amino acid is required for maintaining structure or/and function, but are likely to be tolerated at other sites. Consequently, missense variation within a healthy population can mirror the effects of negative selection on protein structure and function, such that functional sites on proteins are often depleted of missense variants. Advances in high-throughput sequencing have dramatically increased the sample size of available human variation data, allowing for population-wide analysis of selective pressures. In this study, we developed a convenient set of tools, called 1D-to-3D, for visualizing the positions of missense variants on protein sequences and structures. We used these tools to characterize human homologues of the ARID family of gene regulators. ARID family members are implicated in multiple cancer types, developmental disorders, and immunological diseases but current understanding of their mechanistic roles is incomplete. Combined with phylogenetic and structural analyses, our approach allowed us to characterise sites important for protein-protein interactions, histone modification recognition, and DNA binding by the ARID proteins. We find that comparing missense depletion patterns among paralogs can reveal sub-functionalization at the level of domains. We propose that visualizing missense variants and their depletion on structures can serve as a valuable tool for complementing evolutionary and experimental findings.

Isolation of Nucleoid Fraction from Mycoplasma gallisepticum Cells with Synchronized Cell Division.

It is assumed that unknown mechanisms can be involved in adaptation Mycoplasma gallisepticum to unfavorable factors, one of these can be local rearrangements of the structure and spatial organization of the chromosome. To study these mechanisms, we obtained a culture of M. gallisepticum with synchronized division and isolated the nucleoid fraction from this culture by the method of mild cell lysis and centrifugation in a sucrose gradient. Liquid chromatography-mass spectrometry analysis of the proteome showed that in comparison with the cell lysate, the nucleoid fraction was enriched with DNA-binding proteins. This analysis will help to find new nucleoid-associated proteins and to study their dynamics, distribution, and their role during infection and under stress conditions.

Classification of MSH6 Variants of Uncertain Significance Using Functional Assays.

Lynch syndrome (LS) is one of the most common hereditary cancer predisposition syndromes worldwide. Individuals with LS have a high risk of developing colorectal or endometrial cancer, as well as several other cancers. LS is caused by autosomal dominant pathogenic variants in one of the DNA mismatch repair (MMR) genes MLH1, MSH2, PMS2 or MSH6, and typically include truncating variants, such as frameshift, nonsense or splicing variants. However, a significant number of missense, intronic, or silent variants, or small in-frame insertions/deletions, are detected during genetic screening of the MMR genes. The clinical effects of these variants are often more difficult to predict, and a large fraction of these variants are classified as variants of uncertain significance (VUS). It is pivotal for the clinical management of LS patients to have a clear genetic diagnosis, since patients benefit widely from screening, preventive and personal therapeutic measures. Moreover, in families where a pathogenic variant is identified, testing can be offered to family members, where non-carriers can be spared frequent surveillance, while carriers can be included in cancer surveillance programs. It is therefore important to reclassify VUSs, and, in this regard, functional assays can provide insight into the effect of a variant on the protein or mRNA level. Here, we briefly describe the disorders that are related to MMR deficiency, as well as the structure and function of MSH6. Moreover, we review the functional assays that are used to examine VUS identified in MSH6 and discuss the results obtained in relation to the ACMG/AMP PS3/BS3 criterion. We also provide a compiled list of the MSH6 variants examined by these assays. Finally, we provide a future perspective on high-throughput functional analyses with specific emphasis on the MMR genes.

Bound2Learn: a machine learning approach for classification of DNA-bound proteins from single-molecule tracking experiments.

DNA-bound proteins are essential elements for the maintenance, regulation, and use of the genome. The time they spend bound to DNA provides useful information on their stability within protein complexes and insight into the understanding of biological processes. Single-particle tracking allows for direct visualization of protein-DNA kinetics, however, identifying whether a molecule is bound to DNA can be non-trivial. Further complications arise when tracking molecules for extended durations in processes with slow kinetics. We developed a machine learning approach, termed Bound2Learn, using output from a widely used tracking software, to robustly classify tracks in order to accurately estimate residence times. We validated our approach in silico, and in live-cell data from Escherichia coli and Saccharomyces cerevisiae. Our method has the potential for broad utility and is applicable to other organisms.

An Overview of Dps: Dual Acting Nanovehicles in Prokaryotes with DNA Binding and Ferroxidation Properties.

DNA binding proteins under starvation (Dps) are proteins belonging to the ferritin family with the capacity for DNA binding, in addition to iron storage and ferroxidation. Present only in the prokaryotes, these multifaceted proteins have been assigned with a number of roles, from pathogenesis to nucleoid condensation and protection. They have a significant role in protecting the cells from free radical assaults, indirectly by sequestration of iron and by directly binding to the DNA. Due to their symmetry, stability and biomineralization capacity, these proteins have ever increasing potential applications in biotechnology and drug delivery. This chapter tries to bring together all these aspects of Dps in the view of current understanding and older perspectives by studies of our group as well as other experts in the field.

Redefining the H-NS protein family: a diversity of specialized core and accessory forms exhibit hierarchical transcriptional network integration.

H-NS is a nucleoid structuring protein and global repressor of virulence and horizontally-acquired genes in bacteria. H-NS can interact with itself or with homologous proteins, but protein family diversity and regulatory network overlap remain poorly defined. Here, we present a comprehensive phylogenetic analysis that revealed deep-branching clades, dispelling the presumption that H-NS is the progenitor of varied molecular backups. Each clade is composed exclusively of either chromosome-encoded or plasmid-encoded proteins. On chromosomes, stpA and newly discovered hlpP are core genes in specific genera, whereas hfp and newly discovered hlpC are sporadically distributed. Six clades of H-NS plasmid proteins (Hpp) exhibit ancient and dedicated associations with plasmids, including three clades with fidelity for plasmid incompatibility groups H, F or X. A proliferation of H-NS homologs in Erwiniaceae includes the first observation of potentially co-dependent H-NS forms. Conversely, the observed diversification of oligomerization domains may facilitate stable co-existence of divergent homologs in a genome. Transcriptomic and proteomic analysis in Salmonella revealed regulatory crosstalk and hierarchical control of H-NS homologs. We also discovered that H-NS is both a repressor and activator of Salmonella Pathogenicity Island 1 gene expression, and both regulatory modes are restored by Sfh (HppH) in the absence of H-NS.

A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

Comprehensive Analysis and Functional Studies of WRKY Transcription Factors in Nelumbo nucifera.

The WRKY family is one of the largest transcription factor (TF) families in plants and plays central roles in modulating plant stress responses and developmental processes, as well as secondary metabolic regulations. Lotus (Nelumbo nucifera) is an aquatic crop that has significant food, ornamental and pharmacological values. Here, we performed an overview analysis of WRKY TF family members in lotus, and studied their functions in environmental adaptation and regulation of lotus benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 65 WRKY genes were identified in the lotus genome and they were well clustered in a similar pattern with their Arabidopsis homologs in seven groups (designated I, IIa-IIe, and III), although no lotus WRKY was clustered in the group IIIa. Most lotus WRKYs were functionally paired, which was attributed to the recently occurred whole genome duplication in lotus. In addition, lotus WRKYs were regulated dramatically by salicilic acid (SA), jasmonic acid (JA), and submergence treatments, and two lotus WRKYs, NnWRKY40a and NnWRKY40b, were significantly induced by JA and promoted lotus BIA biosynthesis through activating BIA biosynthetic genes. The investigation of WRKY TFs for this basal eudicot reveals new insights into the evolution of the WRKY family, and provides fundamental information for their functional studies and lotus breeding.

DELAY OF GERMINATION 1-LIKE 4 acts as an inducer of seed reserve accumulation.

More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.

The RAG2 gene of yellow catfish (Tachysurus fulvidraco) and its immune response against Edwardsiella ictaluri infection.

Recombination-activating gene 2 (rag 2) allies with recombination-activating gene 1 (rag 1) and regulates the V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes. Being a key player in the adaptive immune response of vertebrates, functional characterization of rag 2 from yellow catfish is beneficial for understanding the biological response towards the pathogens. In this report, we have cloned and characterized the rag 2 gene of yellow catfish, and a particular pattern of expression was analysed in the major tissues of yellow catfish. The results showed that the open reading frame (ORF) of yellow catfish rag 2 was 1596 bp in length, which encodes a peptide of 531 amino acids. The multiple sequence alignment and phylogenetic analysis of rag 2 of yellow catfish with other species showed the conserved regions and the classical taxonomic evolution among the different vertebrate species. The qRT-PCR and Western blot results revealed that rag 2 transcripts and proteins were present in various tissues of yellow catfish with relatively high expression in the tissues of the thymus, head-kidney, and spleen. The systematic distribution analysis of the rag 2 expression by immunohistochemistry (IHC) using the rabbit polyclonal antibody, exposed relatively high expression in head kidney, spleen and thymus tissues after infected with Edwardsiella ictaluri. Moreover, the temporal expression of rag 2 and pro-inflammatory cytokines (IL-1ß and TNF-α) were significantly upregulated at different time points in the specific lymphoid tissues of yellow catfish following E. ictaluri infection. Our findings suggest that rag 2 potentially exhibited the immunological response in primary lymphoid tissues of yellow catfish against bacterial infection. This study will provide an essential source about rag 2 gene and its relationship with the inflammatory cytokines during infection.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

Effective DNA binding protein prediction by using key features via Chou's general PseAAC.

DNA-binding proteins (DBPs) are responsible for several cellular functions, starting from our immunity system to the transport of oxygen. In the recent studies, scientists have used supervised machine learning based methods that use information from the protein sequence only to classify the DBPs. Most of the methods work effectively on the train sets but performance of most of them degrades in the independent test set. It shows a room for improving the prediction method by reducing over-fitting. In this paper, we have extracted several features solely using the protein sequence and carried out two different types of feature selection on them. Our results have proven comparable on training set and significantly improved on the independent test set. On the independent test set our accuracy was 82.26% which is 1.62% improved compared to the previous best state-of-the-art methods. Performance in terms of sensitivity and area under receiver operating characteristic curve for the independent test set was also higher and they were 0.95 and 0.823 respectively.

Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs.

BACKGROUND: Our understanding of eukaryotic gene regulation is limited by the complexity of protein-DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease cleavage method that profiles DNA-binding proteins, histones and chromatin-modifying proteins in situ with exceptional sensitivity and resolution. RESULTS: Here, we describe an automated CUT&RUN platform and apply it to characterize the chromatin landscapes of human cells. We find that automated CUT&RUN profiles of histone modifications crisply demarcate active and repressed chromatin regions, and we develop a continuous metric to identify cell-type-specific promoter and enhancer activities. We test the ability of automated CUT&RUN to profile frozen tumor samples and find that our method readily distinguishes two pediatric glioma xenografts by their subtype-specific gene expression programs. CONCLUSIONS: The easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples.

Rag1 and rag2 gene expressions identify lymphopoietic tissues in juvenile and adult Chinese giant salamander (Andrias davidianus).

Rag1 and rag2 are two closely linked recombination activating genes required for V(D)J recombination of antigen receptors in immature lymphocytes, whose expression can serve as marker to identify the lymphopoietic tissues. To study the development of lymphopoietic tissues in Chinese giant salamander (Andrias davidianus), the Chinese giant salamander rag1 and rag2 coding sequences were cloned and determined. High transcript levels of rag1 and rag2 were co-detected in the thymus before 14 months of age, whereas levels were lower in spleen, liver and kidney at all stage of development. The spatial expression patterns of rag1 and rag2 were studied in combination with igY and tcrß gene expression using in situ hybridization. Significant transcript signals for rag1, rag2, tcrß and igY were detected not only in the thymus and spleen but also the liver and kidney of juvenile and adult Chinese giant salamanders, which suggests that cells of lymphocyte lineage are present in multiple tissues of the Chinese giant salamander. This implies that lymphopoiesis may take place in these tissues. The tissue morphology of thymus suggested that the branched thymic primordium developed into mature organ with the development of thymocyte from juvenile to adult. These results not only confirm that as expected the thymus and spleen are primordial lymphopoietic tissues but also suggest that the liver and kidney provide site of lymphocyte differentiation in Chinese giant salamander.

Caught with One's Zinc Fingers in the Genome Integrity Cookie Jar.

Zinc finger (ZnF) domains are present in at least 5% of human proteins. First characterized as binding to DNA, ZnFs display extraordinary binding plasticity and can bind to RNA, lipids, proteins, and protein post-translational modifications (PTMs). The diverse binding properties of ZnFs have made their functional characterization challenging. While once confined to large and poorly characterized protein families, proteomic, cellular, and molecular studies have begun to shed light on their involvement as protectors of the genome. We focus here on the emergent roles of ZnF domain-containing proteins in promoting genome integrity, including their involvement in telomere maintenance and DNA repair. These findings have highlighted the need for further characterization of ZnF proteins, which can reveal the functions of this large gene class in normal cell function and human diseases, including those involving genome instability such as aging and cancer.

Underground cryptic speciation within the Maghreb: Multilocus phylogeography sheds light on the diversification of the checkerboard worm lizard Trogonophis wiegmanni.

Biogeographic and evolutionary patterns in the North African portion of the Western Palaearctic are poorly known. A high fraction of undescribed diversity is expected in this region, especially in groups such as reptiles. Here we used mitochondrial (12S, 16S, cytb) and nuclear (pomc, rag2, cmos) markers and morphological data to investigate phyletic diversification and phylogeographical structure in the amphisbaenian Trogonophis wiegmanni endemic to the Maghreb. Phylogenetic and molecular dating analyses based on gene trees and species trees support three deeply divergent lineages of Pliocene origin, two in Morocco and one in central Algeria and Tunisia. Parapatry, reciprocal monophyly, high genetic divergence and limited morphological differentiation between them suggest that these lineages represent independent cryptic taxonomic units. Emerging lines of evidence from this study and from available literature on Maghreb taxa support (i) a major biogeographic break between western and eastern Maghreb and (ii) a role of the Atlas as a biogeographic divide within the western Maghreb (Morocco). The origin of these biogeographic units is probably associated with the evolutionary events prompted by the Late Miocene palaeogeographic setting and later by Plio-Pleistocene climatic changes and their interplay with prominent orographic barriers within North Africa.

Growth phase-dependent expression profiles of three vital H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 and on the pCAR1 plasmid.

BACKGROUND: H-NS family proteins are nucleoid-associated proteins that form oligomers on DNA and function as global regulators. They are found in both bacterial chromosomes and plasmids, and were suggested to be candidate effectors of the interaction between them. TurA and TurB are the predominantly expressed H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440, while Pmr is encoded on the carbazole-degradative incompatibility group P-7 plasmid pCAR1. Previous transcriptome analyses suggested that they function cooperatively, but play different roles in the global transcriptional network. In addition to differences in protein interaction and DNA-binding functions, cell expression levels are important in clarifying the detailed underlying mechanisms. Here, we determined the precise protein amounts of TurA, TurB, and Pmr in KT2440 in the presence and absence of pCAR1. RESULTS: The intracellular amounts of TurA and TurB in KT2440 and KT2440(pCAR1) were determined by quantitative western blot analysis using specific antibodies. The amount of TurA decreased from the log phase (~80,000 monomers per cell) to the stationary phase (~20,000 monomers per cell), while TurB was only detectable upon entry into the stationary phase (maximum 6000 monomers per cell). Protein amounts were not affected by pCAR1 carriage. KT2440(pCAR1pmrHis), where histidine-tagged Pmr is expressed under its original promotor, was used to determine the intracellular amount of Pmr, which was constant (~30,000 monomers per cell) during cell growth. Quantitative reverse transcription PCR demonstrated that the transcriptional levels of turA and turB were consistent with protein expression, though the transcriptional and translational profiles of Pmr differed. CONCLUSION: The amount of TurB increases as TurA decreases, and the amount of Pmr does not affect the amounts of TurA and TurB. This is consistent with our previous observation that TurA and TurB play complementary roles, whereas Pmr works relatively independently. This study provides insight into the molecular mechanisms underlying reconstitution of the transcriptional network in KT2440 by pCAR1 carriage.

Cloning, characterization and subcellular localization of Nuclear LIM interactor interacting factor gene from Leishmania donovani.

LIM domains are zinc-binding motifs that mediate protein-protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

Regulation of Polyhydroxybutyrate Accumulation in Sinorhizobium meliloti by the Trans-Encoded Small RNA MmgR.

Riboregulation has a major role in the fine-tuning of multiple bacterial processes. Among the RNA players, trans-encoded untranslated small RNAs (sRNAs) regulate complex metabolic networks by tuning expression from multiple target genes in response to numerous signals. In Sinorhizobium meliloti, over 400 sRNAs are expressed under different stimuli. The sRNA MmgR (standing for Makes more granules Regulator) has been of particular interest to us since its sequence and structure are highly conserved among the alphaproteobacteria and its expression is regulated by the amount and quality of the bacterium's available nitrogen source. In this work, we explored the biological role of MmgR in S. meliloti 2011 by characterizing the effect of a deletion of the internal conserved core of mmgR (mmgRΔ33-51). This mutation resulted in larger amounts of polyhydroxybutyrate (PHB) distributed into more intracellular granules than are found in the wild-type strain. This phenotype was expressed upon cessation of balanced growth owing to nitrogen depletion in the presence of surplus carbon (i.e., at a carbon/nitrogen molar ratio greater than 10). The normal PHB accumulation was complemented with a wild-type mmgR copy but not with unrelated sRNA genes. Furthermore, the expression of mmgR limited PHB accumulation in the wild type, regardless of the magnitude of the C surplus. Quantitative proteomic profiling and quantitative reverse transcription-PCR (qRT-PCR) revealed that the absence of MmgR results in a posttranscriptional overexpression of both PHB phasin proteins (PhaP1 and PhaP2). Together, our results indicate that the widely conserved alphaproteobacterial MmgR sRNA fine-tunes the regulation of PHB storage in S. melilotiIMPORTANCE High-throughput RNA sequencing has recently uncovered an overwhelming number of trans-encoded small RNAs (sRNAs) in diverse prokaryotes. In the nitrogen-fixing alphaproteobacterial symbiont of alfalfa root nodules Sinorhizobium meliloti, only four out of hundreds of identified sRNA genes have been functionally characterized. Thus, uncovering the biological role of sRNAs currently represents a major issue and one that is particularly challenging because of the usually subtle quantitative regulation contributed by most characterized sRNAs. Here, we have characterized the function of the broadly conserved alphaproteobacterial sRNA gene mmgR in S. meliloti Our results strongly suggest that mmgR encodes a negative regulator of the accumulation of polyhydroxybutyrate, the major carbon and reducing power storage polymer in S. meliloti cells growing under conditions of C/N overbalance.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. The information content inherent in this modification alters the affinity and the specificity of DNA binding; some proteins favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events which impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the C2H2 zinc finger domain, and (3) the SET- and RING finger-associated (SRA) domain. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and this difference in the recognition mechanism renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this review, we summarize structural and biochemical analyses elucidating the mechanism for recognition of DNA methylation and correlate this information with emerging genomic and functional data.