Two-hybrid Method Research Articles

Characterization of a Sea Urchin IQ Motif Containing Protein D as a Coactivator of Nuclear Receptors.

Nuclear receptor (NR) interacting proteins, such as coactivators and corepressors, play a crucial role in specifying the transcriptional activity of the receptor. However, little is known about the functional features of the NR coregulators in marine invertebrates. Using the yeast two-hybrid screening method, a sea urchin oocyte cDNA library was screened for proteins that interact with the ligand-binding domain of human RXRα (hRXRα) as the bait protein in the presence of 9-cis retinoic acid. Here, we describe IQ motif containing protein D (IQCD) as an RXR-interacting coactivator. The open reading frame of Strongylocentrotus nudus IQCD (SnIQCD) cDNA contains 1464 bp encoding a protein of 487 amino acids. SnIQCD and the vertebrate IQCDs contain well-conserved C-terminal IQ motifs and coiled-coil domains. The interactions between RXRα and IQCD were confirmed by an immunoprecipitation assay and a mammal two-hybrid assay. RXRα preferentially interacted with the C-terminal half including IQ motif than the N-terminal half of SnIQCD. The coactivator interacting LXXLL motif in SnIQCD is not directly involved in the interaction with RXRα. SnIQCD overexpression increased the basal RXR transactivation of a RXR-responsive reporter gene. Furthermore, SnIQCD enhanced the transcriptional activity of RXR heterodimeric partners such as RAR, PPAR, and the steroid hormone receptor family members from mammals, teleost fish, and sea urchin. Taken together, we suggest that IQCD orthologs are able to function as transcriptional coactivators cooperating with NRs.

MdHIR proteins repress anthocyanin accumulation by interacting with the MdJAZ2 protein to inhibit its degradation in apples

In higher plants, jasmonate ZIM-domain (JAZ) proteins negatively regulate the biosynthesis of anthocyanins by interacting with bHLH transcription factors. However, it is largely unknown if and how other regulators are involved in this process. In this study, the apple MdJAZ2 protein was characterized in regards to its function in the negative regulation of anthocyanin accumulation and peel coloration. MdJAZ2 was used as a bait to screen a cDNA library using the yeast two-hybrid method. The hypersensitive induced reaction (HIR) proteins, MdHIR2 and MdHIR4, were obtained from this yeast two-hybrid. The ZIM domain of MdJAZ2 and the PHB domain of the MdHIR proteins are necessary for their interactions. The interactions were further verified using an in vitro pull-down assay. Subsequently, immunoblotting assays demonstrated that MdHIR4 enhanced the stability of the MdJAZ2-GUS protein. Finally, a viral vector-based transformation method showed that MdHIR4 inhibited anthocyanin accumulation and fruit coloration in apple by modulating the expression of genes associated with anthocyanin biosynthesis.

LMO2 Enhances Lamellipodia/Filopodia Formation in Basal-Type Breast Cancer Cells by Mediating ARP3-Profilin1 Interaction.

BackgroundThe human LMO2 gene was first cloned from an acute T lymphocytic leukemia patient; it is primarily expressed in hematopoietic and vascular endothelial systems, and functions as a pivotal transcriptional regulator during embryonic hematopoiesis and angiogenesis. However, some recent reports indicated that LMO2 is widely expressed in many tissues and tumors, predominantly in cytoplasm, and revealed complicated functions on tumor behaviors in a variety of cancer types. As an adaptor molecule, binding partners and function details of LMO2 in these solid tumors need to be further investigated.Material/MethodsIn this study, we used yeast two-hybrid method to screen potential LMO2 interacting partners, MBP-pulldown, and co-immunoprecipitation assay to confirm protein-protein interactions, and confocal microscopy to reveal the subcellular localization of relevant proteins and actin cytoskeleton changes in relevant cells.ResultsWe found that ARP3 and profilin1 were 2 binding partners of LMO2, primarily in cytoplasm. LMO2. Functionally, LMO2 mediated the assembly of a complex including ARP3, profilin1, and actin monomer, increased actin monomer binding to profilin1, and promoted lamellipodia/filopodia formation in basal-type breast cancer cells.ConclusionsOur data indicate a novel functional mechanism of LMO2 in facilitating the delivery of actin monomers to the branched microfilament and increasing lamellipodia/filopodia formation in basal-type breast cancer cells, suggesting a cancer-promoting role of LMO2 in a subtype-dependent manner and its potential as a subtype-specific biomarker for clinical treatment of breast cancers.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders.

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution.

SRYTH: A New Yeast Two-Hybrid Method.

Many biological processes are regulated by protein-protein interactions, and the analysis of these interactions has been a productive endeavor contributing to our understanding of cellular organization and function. The yeast two-hybrid technique is a widely used, powerful method of analyzing protein-protein interactions. The currently used formats, however, have inherent limitations, providing an opportunity to develop new alternatives that extend our ability to detect protein-protein interactions of biological relevance. Here we present a two-hybrid system named SRYTH (Ste11p/Ste50p related yeast two-hybrid) based on the Ste11p/Ste50p interaction that uses the activation of the HOG pathway of Saccharomyces cerevisiae as a reporter for interactions. The system is suitable for detecting cytoplasmic protein interactions in their natural subcellular environment, and has been successfully used to investigate protein-protein interactions, including transcription factor associations, in Candida albicans.

Prediction of Intra-Species Protein-Protein Interactions in Enteropathogens Facilitating Systems Biology Study

Protein-protein interactions in Escherichia coli (E. coli) has been studied extensively using high throughput methods such as tandem affinity purification followed by mass spectrometry and yeast two-hybrid method. This can in turn be used to understand the mechanisms of bacterial cellular processes. However, experimental characterization of such huge amount of interactions data is not available for other important enteropathogens. Here, we propose a support vector machine (SVM)-based prediction model using the known PPIs data of E. coli that can be used to predict PPIs in other enteropathogens, such as Vibrio cholerae, Salmonella Typhi, Shigella flexneri and Yersinia entrocolitica. Different features such as domain-domain association (DDA), network topology, and sequence information were used in developing the SVM model. The proposed model using DDA, degree and amino acid composition features has achieved an accuracy of 82% and 62% on 5-fold cross validation and blind E. coli datasets, respectively. The predicted interactions were validated by Gene Ontology (GO) semantic similarity measure and String PPIs database (experimental PPIs only). Finally, we have developed a user-friendly webserver named EnPPIpred to predict intra-species PPIs in enteropathogens, which will be of great help for the experimental biologists. The webserver EnPPIpred is freely available at http://bicresources.jcbose.ac.in/ssaha4/EnPPIpred/.

Regulation of Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) by protocadherin-γC5 (Pcdh-γC5)

Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. It is important to identify an endogenous regulator of CaMKP. Using an Escherichia coli two-hybrid screening method, we identified the C-terminal cytoplasmic fragment of protocadherin γ subfamily C5 (Pcdh-γC5), which was generated by intracellular processing, as a CaMKP-binding protein. Dephosphorylation of phosphorylated Ca2+/calmodulin-dependent protein kinase I (CaMKI) by CaMKP was significantly activated by the C-terminal cytoplasmic fragment, Pcdh-γC5(715–944), both in vitro and in cells, suggesting that the C-terminal fragment functions as an endogenous activator of CaMKP. The nuclear translocation of the fragment was blocked by its binding to cytoplasmic CaMKP to form a ternary complex with CaMKI. Taken together, these results strongly suggest that the C-terminal cytoplasmic fragment of Pcdh-γC5 acts as a scaffold for CaMKP and CaMKI to regulate CaMKP activity. These findings may provide new insights into the reversible regulation of CaMKP in cells.

Genome-wide prediction of prokaryotic two-component system networks using a sequence-based meta-predictor.

BackgroundTwo component systems (TCS) are signalling complexes manifested by a histidine kinase (receptor) and a response regulator (effector). They are the most abundant signalling pathways in prokaryotes and control a wide range of biological processes. The pairing of these two components is highly specific, often requiring costly and time-consuming experimental characterisation. Therefore, there is considerable interest in developing accurate prediction tools to lessen the burden of experimental work and cope with the ever-increasing amount of genomic information.ResultsWe present a novel meta-predictor, MetaPred2CS, which is based on a support vector machine. MetaPred2CS integrates six sequence-based prediction methods: in-silico two-hybrid, mirror-tree, gene fusion, phylogenetic profiling, gene neighbourhood, and gene operon. To benchmark MetaPred2CS, we also compiled a novel high-quality training dataset of experimentally deduced TCS protein pairs for k-fold cross validation, to act as a gold standard for TCS partnership predictions. Combining individual predictions using MetaPred2CS improved performance when compared to the individual methods and in comparison with a current state-of-the-art meta-predictor.ConclusionWe have developed MetaPred2CS, a support vector machine-based metapredictor for prokaryotic TCS protein pairings. Central to the success of MetaPred2CS is a strategy of integrating individual predictors that improves the overall prediction accuracy, with the in-silico two-hybrid method contributing most to performance. MetaPred2CS outperformed other available systems in our benchmark tests, and is available online at http://metapred2cs.ibers.aber.ac.uk, along with our gold standard dataset of TCS interaction pairs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0741-7) contains supplementary material, which is available to authorized users.

Banana Ovate family protein MaOFP1 and MADS-box protein MuMADS1 antagonistically regulated banana fruit ripening.

The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.

Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis.

Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins). Incs represent 7–10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH) method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF, and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

MAPPIT, a mammalian two-hybrid method for in-cell detection of protein-protein interactions.

MAPPIT (MAmmalian Protein-Protein Interaction Trap) is a two-hybrid technology that facilitates the detection and analysis of interactions between proteins in living mammalian cells. The system is based on type 1 cytokine receptor signaling. The bait protein of interest is fused to a chimeric signaling-deficient cytokine receptor, the signaling competence of which is restored upon recruitment of a prey protein that is coupled to a functional cytokine receptor domain. MAPPIT exhibits an excellent signal-to-noise ratio, detects a wide variety of protein-protein interactions (PPIs) including transient and indirect interactions, and has been shown to be highly complementary to other two-hybrid methods with respect to the interactions it can detect. Variants of the method were developed to allow large-scale PPI screening, mapping of protein interaction interfaces, PPI inhibitor screening and drug profiling. This chapter describes a basic 4-day MAPPIT protocol for the analysis of interaction between two designated proteins.

Reverse two-hybrid techniques in the yeast Saccharomyces cerevisiae.

Use of the yeast two-hybrid system has provided definition to many previously uncharacterized pathways through the identification and characterization of novel protein-protein interactions. The two-hybrid system uses the bifunctional nature of transcription factors, such as the yeast enhancer Gal4, to allow protein-protein interactions to be monitored through changes in transcription of reporter genes. Once a positive interaction has been identified, either of the interacting proteins can be mutated by site-specific or randomly introduced changes, to produce proteins with a decreased ability to interact. Mutants generated using this strategy are very powerful reagents in tests of the biological significance of the interaction and in defining the residues involved in the interaction. Such techniques are termed reverse two-hybrid methods. We describe a reverse two-hybrid method that generates loss-of-interaction mutations of the catalytic subunit of the Escherichia coli heat-labile toxin (LTA1) with decreased binding to the active (GTP-bound) form of human ARF3, its protein cofactor. While newer methods are emerging for performing interaction screens in mammalian cells, instead of yeast, the use of reverse two-hybrid in yeast remains a robust and powerful means of identifying loss-of-interaction point mutants and compensating changes that remain among the most powerful tools of testing the biological significance of a protein-protein interaction.

The E3 Ubiquitin Ligase Thyroid Hormone Receptor-interacting Protein 12 Targets Pancreas Transcription Factor 1a for Proteasomal Degradation

Pancreas transcription factor 1a (PTF1a) plays a crucial role in the early development of the pancreas and in the maintenance of the acinar cell phenotype. Several transcriptional mechanisms regulating expression of PTF1a have been identified. However, regulation of PTF1a protein stability and degradation is still unexplored. Here, we report that inhibition of proteasome leads to elevated levels of PTF1a and to the existence of polyubiquitinated forms of PTF1a. We used the Sos recruitment system, an alternative two-hybrid system method to detect protein-protein interactions in the cytoplasm and to map the interactome of PTF1a. We identified TRIP12 (thyroid hormone receptor-interacting protein 12), an E3 ubiquitin-protein ligase as a new partner of PTF1a. We confirmed PTF1a/TRIP12 interaction in acinar cell lines and in co-transfected HEK-293T cells. The protein stability of PTF1a is significantly increased upon decreased expression of TRIP12. It is reduced upon overexpression of TRIP12 but not a catalytically inactive TRIP12-C1959A mutant. We identified a region of TRIP12 required for interaction and identified lysine 312 of PTF1a as essential for proteasomal degradation. We also demonstrate that TRIP12 down-regulates PTF1a transcriptional and antiproliferative activities. Our data suggest that an increase in TRIP12 expression can play a part in PTF1a down-regulation and indicate that PTF1a/TRIP12 functional interaction may regulate pancreatic epithelial cell homeostasis.

Proteins affecting thylakoid morphology – the key to understanding vesicle transport in chloroplasts?

We recently showed that a Rab protein, CPRabA5e (CP = chloroplast localized), is located in chloroplasts of Arabidopsis thaliana where it is involved in various processes, such as thylakoid biogenesis and vesicle transport. Using a yeast two-hybrid method, CPRabA5e was shown to interact with a number of chloroplast proteins, including the CURVATURE THYLAKOID 1A (CURT1A) protein and the light-harvesting chlorophyll a/b binding protein (LHCB1.5). CURT1A has recently been shown to modify thylakoid architecture by inducing membrane curvature in grana, whereas LHCB1.5 is a protein of PSII (Photosystem II) facilitating light capture. LHCB1.5 is imported to chloroplasts and transported to thylakoid membranes using the post-translational Signal Recognition Particle (SRP) pathway. With this information as starting point, we here discuss their subsequent protein-protein interactions, given by the literature and Interactome 3D. CURT1A itself and several of the proteins interacting with CURT1A and LHCB1.5 have relations to vesicle transport and thylakoid morphology, which are also characteristics of cprabA5e mutants. This highlights the previous hypothesis of an alternative thylakoid targeting pathway for LHC proteins using vesicles, in addition to the SRP pathway.

The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro

The nonstructural protein 9 (Nsp9) of porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized to play important roles in viral replication. The present study first screened that the DEAD-box RNA helicase 5 (DDX5) was a cellular protein interacting with the Nsp9 of PRRSV by a yeast two-hybrid method in a pulmonary alveolar macrophages (PAMs) cDNA library. Next, DDX5 was shown to interact with viral Nsp9 in the co-transfected HEK293 cells with the DDX5- and Nsp9-expressing plasmids, and the interaction between endogenous DDX5 and Nsp9 was also confirmed in MARC-145 cells infected with the Nsp9-expressing lentiviruses. Then, the interacting domains between DDX5 and Nsp9 were determined to be the DEXDc and HELICc domains in DDX5 and the RdRp domain in Nsp9, respectively. Moreover, in the HEK293 cells, MARC-145 cells and PAM cell lines co-transfected with the DDX5- and Nsp9-expressing plasmids, Nsp9 was shown to co-localize with DDX5 in the cytoplasm with a perinuclear pattern, and meanwhile in PRRSV-infected MARC-145 cells and PAMs, endogenous DDX5 was also found to co-localize with Nsp9. Finally, silencing the DDX5 gene in MARC-145 cells significantly impacted the replication of PRRSV, and while the over-expression of DDX5 could slightly enhance viral replication. These findings indicate that DDX5 positively regulates the replication of PRRSV via its interaction with viral Nsp9 in vitro.

Involvement of potato (Solanum tuberosum L.) MKK6 in response to potato virus Y.

Mitogen-activated protein kinase (MAPK) cascades have crucial roles in the regulation of plant development and in plant responses to stress. Plant recognition of pathogen-associated molecular patterns or pathogen-derived effector proteins has been shown to trigger activation of several MAPKs. This then controls defence responses, including synthesis and/or signalling of defence hormones and activation of defence related genes. The MAPK cascade genes are highly complex and interconnected, and thus the precise signalling mechanisms in specific plant–pathogen interactions are still not known. Here we investigated the MAPK signalling network involved in immune responses of potato (Solanum tuberosum L.) to Potato virus Y, an important potato pathogen worldwide. Sequence analysis was performed to identify the complete MAPK kinase (MKK) family in potato, and to identify those regulated in the hypersensitive resistance response to Potato virus Y infection. Arabidopsis has 10 MKK family members, of which we identified five in potato and tomato (Solanum lycopersicum L.), and eight in Nicotiana benthamiana. Among these, StMKK6 is the most strongly regulated gene in response to Potato virus Y. The salicylic acid treatment revealed that StMKK6 is regulated by the hormone that is in agreement with the salicylic acid-regulated domains found in the StMKK6 promoter. The involvement of StMKK6 in potato defence response was confirmed by localisation studies, where StMKK6 accumulated strongly only in Potato-virus-Y-infected plants, and predominantly in the cell nucleus. Using a yeast two-hybrid method, we identified three StMKK6 targets downstream in the MAPK cascade: StMAPK4_2, StMAPK6 and StMAPK13. These data together provide further insight into the StMKK6 signalling module and its involvement in plant defence.

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.

Elucidation of the specific formation of homo- and heterodimeric forms of ThbZIP1 and its role in stress.

Protein–protein interactions are important for the molecular understanding of the biological processes of proteins. The dimerization of bZIPs (basic leucine zipper proteins) is involved in modifying binding site specificities, altering dimer stability, and permitting a new set of specific protein-to-protein interactions to occur at the promoter. In the present study, we studied the whether ThbZIP1 form homo- and heterodimers using the yeast two-hybrid method. Five bZIP genes were cloned from Tamarix hispida to investigate their interaction with ThbZIP1. Our results showed that ThbZIP1 can form homodimers with itself, and three out of five bZIPs could interact with the ThbZIP1 protein to form heterodimers. Real-time RT-PCR results suggested that these ThbZIPs can all respond to abiotic stresses and abscisic acid (ABA), and shared very similar expression patterns in response to NaCl, ABA or PEG6000. Subcellular localization studies showed that all ThbZIPs are targeted to the nucleus. Our results showed that ThbZIP1 are dimeric proteins, which can form homo- or heterodimers.

Physical Interaction between Bacterial Heat Shock Protein (Hsp) 90 and Hsp70 Chaperones Mediates Their Cooperative Action to Refold Denatured Proteins

In eukaryotes, heat shock protein 90 (Hsp90) is an essential ATP-dependent molecular chaperone that associates with numerous client proteins. HtpG, a prokaryotic homolog of Hsp90, is essential for thermotolerance in cyanobacteria, and in vitro it suppresses the aggregation of denatured proteins efficiently. Understanding how the non-native client proteins bound to HtpG refold is of central importance to comprehend the essential role of HtpG under stress. Here, we demonstrate by yeast two-hybrid method, immunoprecipitation assays, and surface plasmon resonance techniques that HtpG physically interacts with DnaJ2 and DnaK2. DnaJ2, which belongs to the type II J-protein family, bound DnaK2 or HtpG with submicromolar affinity, and HtpG bound DnaK2 with micromolar affinity. Not only DnaJ2 but also HtpG enhanced the ATP hydrolysis by DnaK2. Although assisted by the DnaK2 chaperone system, HtpG enhanced native refolding of urea-denatured lactate dehydrogenase and heat-denatured glucose-6-phosphate dehydrogenase. HtpG did not substitute for DnaJ2 or GrpE in the DnaK2-assisted refolding of the denatured substrates. The heat-denatured malate dehydrogenase that did not refold by the assistance of the DnaK2 chaperone system alone was trapped by HtpG first and then transferred to DnaK2 where it refolded. Dissociation of substrates from HtpG was either ATP-dependent or -independent depending on the substrate, indicating the presence of two mechanisms of cooperative action between the HtpG and the DnaK2 chaperone system.

The PpCMT chromomethylase affects cell growth and interacts with the homolog of LIKE HETEROCHROMATIN PROTEIN 1 in the moss Physcomitrella patens

Chromomethylases (CMTs) are plant-specific cytosine DNA methyltransferases that are involved in maintenance of CpNpG methylation. In seed plants, histone methylation and interaction of CMT with LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is essential for recruitment of CMT to target sites. LHP1 has been characterized as a putative component of the POLYCOMB REPRESSIVE COMPLEX1 (PRC1) in plants, and functions downstream of PRC2 to maintain genes in repressed state for orchestrated development. In the present study, we show that targeted disruption of PpCMT results in an approximately 50% reduction in global cytosine methylation levels. This affects growth of apical cells, predominantly growth of side branch initials emerging from chloronema cells. In some places, these cells develop thick walls with plasmolyzed cellular contents. Transcript accumulation patterns of genes involved in apical cell extension and metabolism of hemicelluloses, such as xyloglucans, in the primary cell walls decreased many fold in ppcmt mutant lines, as determined by real-time PCR. Using yeast two-hybrid method and bimolecular fluorescence complementation assay, we show that PpCMT and PpLHP1 interact through their chromo domains, while PpLHP1 homodimerizes through its chromo shadow domain. The results presented in this study provide insight into the role of the single chromomethylase, PpCMT, in proliferation of protonema filaments, and shed light on the evolutionary conservation of proteins interacting with these methylases in the early land plant, Physcomitrella patens.