Cell Migration from Baby to Mother

Fetal cells migrate into the mother during pregnancy. Fetomaternal transfer probably occurs in all pregnancies and in humans the fetal cells can persist for decades. Microchimeric fetal cells are found in various maternal tissues and organs including blood, bone marrow, skin and liver. In mice, fetal cells have also been found in the brain. The fetal cells also appear to target sites of injury. Fetomaternal microchimerism may have important implications for the immune status of women, influencing autoimmunity and tolerance to transplants. Further understanding of the ability of fetal cells to cross both the placental and blood-brain barriers, to migrate into diverse tissues, and to differentiate into multiple cell types may also advance strategies for intravenous transplantation of stem cells for cytotherapeutic repair. Here we discuss hypotheses for how fetal cells cross the placental and blood-brain barriers and the persistence and distribution of fetal cells in the mother.Key Words: fetomaternal microchimerism, stem cells, progenitor cells, placental barrier, blood-brain barrier, adhesion, migrationMicrochimerism is the presence of a small population of genetically distinct and separately derived cells within an individual. This commonly occurs following transfusion or transplantation.^1–3 Microchimerism can also occur between mother and fetus. Small numbers of cells traffic across the placenta during pregnancy. This exchange occurs both from the fetus to the mother (fetomaternal)^4–7 and from the mother to the fetus.^8–10 Similar exchange may also occur between monochorionic twins in utero.^11–13 There is increasing evidence that fetomaternal microchimerism persists lifelong in many child-bearing women.^7,14 The significance of fetomaternal microchimerism remains unclear. It could be that fetomaternal microchimerism is an epiphenomenon of pregnancy. Alternatively, it could be a mechanism by which the fetus ensures maternal fitness in order to enhance its own chances of survival. In either case, the occurrence of pregnancy-acquired microchimerism in women may have implications for graft survival and autoimmunity. More detailed understanding of the biology of microchimeric fetal cells may also advance progress towards cytotherapeutic repair via intravenous transplantation of stem or progenitor cells.Trophoblasts were the first zygote-derived cell type found to cross into the mother. In 1893, Schmorl reported the appearance of trophoblasts in the maternal pulmonary vasculature.¹⁵ Later, trophoblasts were also observed in the maternal circulation.^16–20 Subsequently various other fetal cell types derived from fetal blood were also found in the maternal circulation.^21,22 These fetal cell types included lymphocytes,²³ erythroblasts or nucleated red blood cells,^24,25 haematopoietic progenitors^7,26,27 and putative mesenchymal progenitors.^14,28 While it has been suggested that small numbers of fetal cells traffic across the placenta in every human pregnancy,^29–31 trophoblast release does not appear to occur in all pregnancies.³² Likewise, in mice, fetal cells have also been reported in maternal blood.^33,34 In the mouse, fetomaternal transfer also appears to occur during all pregnancies.³⁵     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Plant pathogenic microbes deliver effector proteins inside host cells to modulate plant defense circuitry and enable parasitic colonization. As genome sequences from plant pathogens become available, genome-wide evolutionary analyses will shed light on how pathogen effector genes evolved and adapted to the cellular environment of their host plants. In the August 2007 issue of Plant Cell, we described adaptive evolution (positive selection) in the cytoplasmic RXLR effectors of three recently sequenced oomycete plant pathogens. Here, we summarize our findings and describe additional data that further validate our approach.Key words: plant-microbe interactions, effectors, gene families, positive selectionA diverse number of plant pathogens, including bacteria, oomycetes, fungi and nematodes, deliver effector proteins inside host cells to modulate plant defense circuitry and enable parasitic colonization.^1–8 Because these so-called cytoplasmic effectors function inside plant cells and produce phenotypes that extend to plant cells and tissues, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host.^9,10 In a study published in the August 2007 issue of Plant Cell, we and our collaborators examined the extent to which positive selection (adaptive evolution) has shaped the evolution of the cytoplasmic effectors of three recently sequenced oomycete plant pathogens Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica (Genome Sequencing Center at Washington University).¹¹     

Tuba and N-WASP function cooperatively to position the central lumen during epithelial cyst morphogenesis

The process of epithelial lumenogenesis requires coordination of a network of signaling machinery communicated to each cell through subsequent cell divisions. Formation of a single hollow lumen has previously been shown to require Tuba, a Cdc42 GEF, for Cdc42 activation and correct spindle orientation. Using a Caco-2 model of lumenogenesis, we show that knockdown (KD) of the actin regulator N-WASP, causes a multilumen phenotype similar to Tuba KD. Defects in lumenogenesis in Tuba KD and N-WASP KD cells are observed at the two-cell stage with inappropriate marking of the pre-apical patch (PAP )—the precursor to lumen formation. Strikingly, both Tuba and N-WASP depend on each other for localization to the PAP. We conclude that N-WASP functions cooperatively with Tuba to facilitate lumenogenesis and this requires the polyproline region of N-WASP.Key words: lumen, N-WASP, tuba, E-cadherin, pre-apical patchMany epithelial tissues are organized as hollow tubes whose open lumina connect the body with its external environment.^1,2 These tubes consist of a monolayer of polarized cells that envelope the central lumen. Lumen formation is thus a key process in epithelial morphogenesis that depends upon cell polarity to establish three cell surface domains: a basal surface adherent to the extracellular matrix, a lateral surface between cells, and an apical surface that is exposed to the luminal fluids. Of note, the apical membrane is biochemically and morphologically distinct from the baso-lateral surfaces and effectively defines the luminal surface.^3,4For a lumen to form, cells must first mark the site at which apical membrane is to be inserted, something that is achieved at the first cell division.⁵ Targeted trafficking of apical membrane constituents defines a pre-apical patch (PAP), the precursor to the definitive lumen.⁵ Such insertion of apical membrane must presumably be coordinated with the assembly of apical junctions to segregate nascent apical from lateral membrane domains.² Subsequent cell divisions direct apical membrane and protein constituents to this point of initial apical membrane placement.⁶ Coordinated luminal positioning enables the initial formation of a single hollow lumen that subsequently expands through polarized fluid secretion to separate apical membranes, such as occurs in the embryonic gastrointestinal tract,⁷ or by apoptosis or autophagy of the central cells as is observed in mammary gland development.^8,9 Failure to establish initial luminal positioning causes defective lumenogenesis, often resulting in multiple, morphologically abnormal lumina.^5,6Crucial to lumenal morphogenesis is then the mechanism(s) that mark the site where the PAP will form. Cdc42 signaling is increasingly implicated in this process,^2,10 with downstream consequences that include control of mitotic spindle orientation,⁵ which itself influences PAP placement⁵ and potentially regulation of cell-cell junctions. Like other Rho family GTPases, the subcellular location of Cdc42 signaling is determined by the action of upstream proteins, notably guanine nucleotide exchange factors (GEFs).^11,12 Of these, Tuba, a Cdc42-specific GEF,¹³ has emerged as a regulator of lumenal morphogenesis that controls PAP placement through mitotic spindle orientation.¹⁰Tuba is also a scaffolding protein¹³ capable of linking the actin assembly machinery with trafficking pathways. Not only is Tuba required for Cdc42 activation to direct spindle orientation,⁵ it also has the potential to interact with phosphoinositides that define the PAP.¹⁴ Additionally, Tuba binds directly to the actin regulator N-WASP, a key molecule in the organization of actin and itself a Cdc42 effector.¹⁵ Further, Tuba and N-WASP cooperate in various forms of actin-driven cellular motility, such as vesicle propulsion and cell invasive behavior.¹⁶ Interestingly, in epithelial cells N-WASP is also found at cadherin-based cell-cell junctions.¹⁷ In fact it has been proposed that N-WASP functions downstream of Tuba in the maintenance of epithelial junctional homeostasis as N-WASP overexpression was capable of rescuing a Tuba KD phenotype.¹⁸ Therefore, Tuba has the potential to play a central role in coordinating the molecular complexes required for productive polarization of epithelial cells and placement of the PAP during lumenogenesis. However, whether other protein interactions contribute to the morphogenetic impact of Tuba remain to be assessed.Three-dimensional cell culture systems are being utilized to identify critical components in lumen formation. In particular, Madin-Darby canine kidney cells (MDCK) and Caco-2 gastrointestinal cells are commonly used to study cyst and/or tubule formation. MDCK cells undergo both cyst and tubule growth, apoptosis being primarily responsible for the final step in lumen formation,¹⁹ while Caco-2 cells primarily utilize fluid influx to expand cysts.⁵ Cyst culture systems replicate aspects of in vivo organogenesis²⁰ providing tangible, powerful models to analyze and dissect the coordinated cellular mechanisms and processes that occur during epithelial morphogenesis.In this study we examined the relationship between Tuba and N-WASP in early epithelial lumenogenesis using Caco-2 three dimensional cyst cultures. Both Tuba and N-WASP RNAi cell lines result in mature cysts with multiple lumina, and at the two-cell stage, formed multiple PAPs. Interestingly, N-WASP KD perturbed Tuba localization at the PAP, however, N-WASP localization to the PAP was not affected to the same extent by Tuba KD. Taken together, these results suggest a complex interrelationship between Tuba and N-WASP for the coordinated formation of a single hollow lumen.     

RALFs: Peptide regulators of plant growth

Peptide signaling regulates a variety of developmental processes and environmental responses in plants.^1–6 For example, the peptide systemin induces the systemic defense response in tomato⁷ and defensins are small cysteine-rich proteins that are involved in the innate immune system of plants.^8,9 The CLAVATA3 peptide regulates meristem size¹⁰ and the SCR peptide is the pollen self-incompatibility recognition factor in the Brassicaceae.^11,12 LURE peptides produced by synergid cells attract pollen tubes to the embryo sac.⁹ RALFs are a recently discovered family of plant peptides that play a role in plant cell growth.Key words: peptide, growth factor, alkalinization     

Indolic secondary metabolites protect Arabidopsis from the oomycete pathogen Phytophthora brassicae

The model plant Arabidopsis thaliana contains a large arsenal of secondary metabolites that are not essential in development but have important ecological functions in counteracting attacks of pathogens and herbivores.^1,2 Preformed secondary compounds are often referred to as phytoanticipins and metabolites, that are synthesized de novo in response to biotic stress are known as phytoalexins.³ Camalexin is the typical phytoalexin of Arabidopsis. It has antimicrobial activity towards some pathogens and was shown to be an important component of disease resistance in several plant pathogen interactions.⁴ Glucosinolates (GS) are characteristic phytoanticipins of the Brassicaceae family including Arabidopsis. GS are best known as repellents or attractants for herbivorous insects and their predators whereas their antimicrobial potential has received relatively little attention.⁵ The GS are glucosides and the biologically active aglycone is released upon biotic stress by glucohydrolase enzymes commenly called myrosinases. Because an Arabidopsis mutant susceptible to the oomycete pathogen Phytophthora brassicae shows a partial deficiency in both camalexin and iGS accumulation we became intrigued by the role of these secondary compounds in disease resistance.^6,7 Our results show that disease resistance of Arabidopsis to P. brassicae is established by the combined action of iGS and camalexin.Key words: Arabidopsis, disease resistance, Phytophthora brassicae, secondary metabolites, indolic glucosinolates, camalexin     

Plant defensins: Defense,development and application

Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins are strongly resistant to fungal pathogens. Based on recent studies, some plant defensins are not merely toxic to microbes but also have roles in regulating plant growth and development.Key words: defensin, antifungal, antimicrobial peptide, development, innate immunityDefensins are diverse members of a large family of cationic host defence peptides (HDP), widely distributed throughout the plant and animal kingdoms.^1–3 Defensins and defensin-like peptides are functionally diverse, disrupting microbial membranes and acting as ligands for cellular recognition and signaling.⁴ In the early 1990s, the first members of the family of plant defensins were isolated from wheat and barley grains.^5,6 Those proteins were originally called γ-thionins because their size (∼5 kDa, 45 to 54 amino acids) and cysteine content (typically 4, 6 or 8 cysteine residues) were found to be similar to the thionins.⁷ Subsequent “γ-thionins” homologous proteins were indentified and cDNAs were cloned from various monocot or dicot seeds.⁸ Terras and his colleagues⁹ isolated two antifungal peptides, Rs-AFP1 and Rs-AFP2, noticed that the plant peptides'' structural and functional properties resemble those of insect and mammalian defensins, and therefore termed the family of peptides “plant defensins” in 1995. Sequences of more than 80 different plant defensin genes from different plant species were analyzed.¹⁰ A query of the UniProt database (www.uniprot.org/) currently reveals publications of 371 plant defensins available for review. The Arabidopsis genome alone contains more than 300 defensin-like (DEFL) peptides, 78% of which have a cysteine-stabilized α-helix β-sheet (CSαβ) motif common to plant and invertebrate defensins.¹¹ In addition, over 1,000 DEFL genes have been identified from plant EST projects.¹²Unlike the insect and mammalian defensins, which are mainly active against bacteria,^2,3,10,13 plant defensins, with a few exceptions, do not have antibacterial activity.¹⁴ Most plant defensins are involved in defense against a broad range of fungi.^2,3,10,15 They are not only active against phytopathogenic fungi (such as Fusarium culmorum and Botrytis cinerea), but also against baker''s yeast and human pathogenic fungi (such as Candida albicans).² Plant defensins have also been shown to inhibit the growth of roots and root hairs in Arabidopsis thaliana¹⁶ and alter growth of various tomato organs which can assume multiple functions related to defense and development.⁴     

Plant Parasitic Oomycetes Such as Phytophthora Species Contain Genes Derived from Three Eukaryotic Lineages

Fungi and the oomycetes include several groups of plant pathogenic microbes. Although these two eukaryotic groups are unrelated they have a number of phenotypic similarities suggested to have evolved convergently. We have recently shown that gene transfer events have occurred from fungi to the oomycetes. These gene transfer events appear to be only one part of a complex and chimeric ancestry for the oomycete genome, which has also received genes from a red algal endosymbiont.Key Words: horizontal gene transfer, osmotrophy, phototrophy, biotrophy, endosymbiosis, fungi, Magnaporthe griseaAs genomic sampling increases, a persistent pattern of horizontal gene transfer (HGT) between microbial lineages is becoming evident.^1,2 So far, patterns of horizontal gene transfer have been identified in four main forms: (A) gene transfer between prokaryote lineages, such that a large proportion of many prokaryote genomes are likely to be chimeric,^3,4 (B) gene transfer from the prokaryote progenitors of the mitochondrion and the plastid organelles to a host eukaryote nuclear genome (e.g., refs. ^5–7), (C) gene transfer from prokaryote genomes to eukaryote microbes, often involving phagocytic eukaryotes and microbes that share similar habitats⁸ and (D) gene transfer from a eukaryotic endosymbiont to their host eukaryotic genomes.^9,10 This fourth form of gene transfer includes secondary and tertiary endosymbiotic events and has so far provided our best examples of eukaryote-to-eukaryote gene transfer.^11,12 Secondary and tertiary endosymbiotic events are typified by the engulfment of a photosynthetic eukaryote by another eukaryote followed by the reduction of the consumed photosynthetic eukaryote and transfer of genes from the endosymbiont to the host nuclei with some retargeting of the transferred gene products back to the remnant organelle.^9,10Gene transfer events can be identified using phylogenetic analysis when an individual gene tree topology contradicts a known species relationship. HGT can only be seriously considered, however, if the gene phylogeny shows that the putative HGT is nested within a donor clade with strong bootstrap support.² Endosymbiosis typically leads to multiple cases of nuclear-encoded genes demonstrating endosymbiotic ancestry, with the candidate genes grouping within a clade representing the lineage that gave rise to the progenitor of the endosymbiont.⁵ There have been multiple cases of both secondary and tertiary endosymbiosis within the eukaryotes, making the evolutionary reconstruction of phototrophy in the eukaryotes highly complex.⁹ Secondary and tertiary endosymbiotic remnant organelles are often identified by the presence of three or more membranes surrounding the organelle body.¹⁰ However, secondary endosymbiotic events have led to a range of different combinations of cell apparatus, from the total loss of the endosymbiont-derived organelle^13,14 to the maintenance of the organelle compartment¹⁰ and the possession of a remnant nucleus as a nucleomorph.¹⁵The oomycetes include the plant pathogenic Phytophthora spp. and are heterokonts (sometimes called Stramenopiles).¹⁶ The heterokonts also encompass numerous groups of photosynthetic algae (e.g., Bolidomonas, Diatoms, Xanthophyceae, Phaeophyceae and Chrysophyceae) and are proposed to be derived from an ancestrally photosynthetic cell that obtained its plastid by engulfment of a red alga.¹⁶ Cytological studies of the oomycetes have so far failed to identify a relic plastid organelle but the recent publication of the Phytophthora sojae and Phytophthora ramorum genomes identified 855 genes putatively originating from the genome of a photosynthetic microbe consistent with a phototrophic ancestry for the oomycetes.¹³Phytophthora plant pathogens include the causal agents of sudden oak death (P. ramorum), potato blight (P. infestans) and, P. sojae which causes serious root and stem rot of soybean plants. Initially, P. infestans was identified as a fungal pathogen and the causal agent of the great 1845 Irish potato famine by Rev. Miles J. Berkeley,¹⁷ due to life cycle similarities and an apparently homologous mode of plant infection to ascomycete plant pathogens. It was only with the use of molecular phylogenetic methods starting with small subunit rDNA analysis¹⁶ followed by multiple concatenated gene phylogenies¹⁸ that the oomycetes were demonstrated to group within the heterokont radiation. With the apparent phylogenetic origins of the oomycetes pinpointed it left the apparent similarities in pathogenic mechanism and infective lifecycle between the filamentous ascomycetes and the oomycetes a mysterious case of convergent evolution.¹⁹During the evolutionary analyses of the predicted proteome of the filamentous plant pathogenic ascomycete Magnaporthe grisea²⁰ we detected a series of unexpected similarities in the genomes of plant pathogenic ascomycetes and the oomycete genomes.¹³ We followed up this observation by further investigation using phylogenetic methods combined with comparative genomic analysis, which revealed a series of HGT events. We subjected our datasets to a range of tests: (A) to test that the level of support for the tree topology seen was robust given random resampling of the sequence alignments used to reconstruct the gene phylogenies; (B) to ensure that the possibility that similar topologies with the oomycete/filamentous ascomycete relationship removed could be rejected at the 0.05 confidence level and; (C) to test for alternative patterns of gene evolution including hidden paralogy (duplication with differential patterns of gene loss) were unlikely. Four of the datasets tested in this way held up to our scrutiny and were thus proposed as fungi-to-oomycete horizontal gene transfers.²¹ The predicted function of three of the four genes (CodB, a purine permease, AraJ, a sugar transporter and a PcaH an extracellular dioxygenase) could conceivably be useful for an osmotrophic microbe living in a plant associated habitat (biotrophy), suggesting that these HGT events could in-part explain the convergently evolved similarities in osmotrophy and filamentous growth habit seen in the oomycetes and fungi. Our analyses also suggested that three of these HGTs originated from a genome closely related to the last common ancestor of the Magnaporthe and Aspergillus evolutionary branches. Although the specific branching position of the transferred lineage could not be pinpointed in the fourth analysis, the same point of origin could not however be excluded. This suggests that the four HGTs we identified could be derived from the same source, a phenomenon similar in pattern (if not involving the same lineages) to that seen for phylogenetic tree topologies used to investigate the endosymbiotic events discussed above. Although these analyses do not shed any light on the circumstances in which these transfers occurred, it is possible that an intimate association between a fungus and a heterokont has led to genetic exchange and demonstrates that eukaryote-to-eukaryote gene transfers are not just associated with the acquisition of phototrophy by secondary/tertiary endosymbiosis.Our published study was conducted using only published genome sequences as a seed for comparative genomic analyses.²¹ However, with the very recent publication of two Phytophthora genomes¹³ it is possible that further analyses will identify additional candidate Phytophthora-Fungi HGT events when they are carried out. These tests may determine how pervasive the pattern of HGT is within the oomycetes.The oomycetes have been classified within the phylum Pseudofungi¹⁶ which comprises a number of microbial lineages with phenotypic similarities to true fungi, including hyphae-like structures and osmotrophy. Originally, the term Pseudofungi was used to group together ‘water-moulds’ possessing mastigonemes (tubular tri-partite hairs) on one flagellum. Currently the phylum Pseudofungi comprises the biotrophic oomycetes including parasites of plants and brown algae, the phagotrophic Developayella and the biotrophic hyphochytrids, including the diatom ectoparasite Pirsonia.¹⁶ It will be interesting to ascertain at what point within the diversification of the Pseudofungi the HGTs that are identified²¹ became fixed and how the acquisition of these phenotypes relates to the evolution of Pseudofungi phenotypes within the heterokonts. Independent of the specific ancestry of the gene transfer events within the Pseudofungi it is clear that P. sojae and P. ramorum have chimeric genomes, originating from three separate eukaryotic lineages, the ancestral heterokont nuclear genome, the red algal endosymbiont and at least four genes of fungal ancestry donated to an oomycete nuclear genome.     

Human non-CG methylation: Are human stem cells plant-like?

Non-CG methylation is well characterized in plants where it appears to play a role in gene silencing and genomic imprinting. Although strong evidence for the presence of non-CG methylation in mammals has been available for some time, both its origin and function remain elusive. In this review we discuss available evidence on non-CG methylation in mammals in light of evidence suggesting that the human stem cell methylome contains significant levels of methylation outside the CG site.Key words: non-CG methylation, stem cells, Dnmt1, Dnmt3a, human methylomeIn plant cells non-CG sites are methylated de novo by Chromomethylase 3, DRM1 and DRM2. Chromomethylase 3, along with DRM1 and DRM2 combine in the maintenance of methylation at symmetric CpHpG as well as asymmetric DNA sites where they appear to prevent reactivation of transposons.¹ DRM1 and DRM2 modify DNA de novo primarily at asymmetric CpH and CpHpH sequences targeted by siRNA.²Much less information is available on non-CG methylation in mammals. In fact, studies on mammalian non-CG methylation form a tiny fraction of those on CG methylation, even though data for cytosine methylation in other dinucleotides, CA, CT and CC, have been available since the late 1980s.³ Strong evidence for non-CG methylation was found by examining either exogenous DNA sequences, such as plasmid and viral integrants in mouse and human cell lines,^4,5 or transposons and repetitive sequences such as the human L1 retrotransposon⁶ in a human embryonic fibroblast cell line. In the latter study, non-CG methylation observed in L1 was found to be consistent with the capacity of Dnmt1 to methylate slippage intermediates de novo.⁶Non-CG methylation has also been reported at origins of replication^7,8 and a region of the human myogenic gene Myf3.⁹ The Myf3 gene is silenced in non-muscle cell lines but it is not methylated at CGs. Instead, it carries several methylated cytosines within the sequence CCTGG. Gene-specific non-CG methylation was also reported in a study of lymphoma and myeloma cell lines not expressing many B lineage-specific genes.¹⁰ The study focused on one specific gene, B29 and found heavy CG promoter methylation of that gene in most cell lines not expressing it. However, in two other cell lines where the gene was silenced, cytosine methylation was found almost exclusively at CCWGG sites. The authors provided evidence suggesting that CCWGG methylation was sufficient for silencing the B29 promoter and that methylated probes based on B29 sequences had unique gel shift patterns compared to non-methylated but otherwise identical sequences.¹⁰ The latter finding suggests that the presence of the non-CG methylation causes changes in the proteins able to bind the promoter, which could be mechanistically related to the silencing seen with this alternate methylation.Non-CG methylation is rarely seen in DNA isolated from cancer patients. However, the p16 promoter region was reported to contain both CG and non-CG methylation in breast tumor specimens but lacked methylation at these sites in normal breast tissue obtained at mammoplasty.¹¹ Moreover, CWG methylation at the CCWGG sites in the calcitonin gene is not found in normal or leukemic lymphocyte DNA obtained from patients.¹² Further, in DNA obtained from breast cancer patients, MspI sites that are refractory to digestion by MspI and thus candidates for CHG methylation were found to carry CpG methylation.¹³ Their resistance to MspI restriction was found to be caused by an unusual secondary structure in the DNA spanning the MspI site that prevents restriction.¹³ This latter observation suggests caution in interpreting EcoRII/BstNI or EcoRII/BstOI restriction differences as due to CWG methylation, since in contrast to the 37°C incubation temperature required for full EcoRII activity, BstNI and BstOI require incubation at 60°C for full activity where many secondary structures are unstable.The recent report by Lister et al.¹⁴ confirmed a much earlier report by Ramsahoye et al.¹⁵ suggesting that non-CG methylation is prevalent in mammalian stem cell lines. Nearest neighbor analysis was used to detect non-CG methylation in the earlier study on the mouse embryonic stem (ES) cell line,¹⁵ thus global methylation patterning was assessed. Lister et al.¹⁴ extend these findings to human stem cell lines at single-base resolution with whole-genome bisulfite sequencing. They report¹⁴ that the methylome of the human H1 stem cell line and the methylome of the induced pluripotent IMR90 (iPS) cell line are stippled with non-CG methylation while that of the human IMR90 fetal fibroblast cell line is not. While the results of the two studies are complementary, the human methylome study addresses locus specific non-CG methylation. Based on that data,¹⁴ one must conclude that non-CG methylation is not carefully maintained at a given site in the human H1 cell line. The average non-CG site is picked up as methylated in about 25% of the reads whereas the average CG methylation site is picked up in 92% of the reads. Moreover, non-CG methylation is not generally present on both strands and is concentrated in the body of actively transcribed genes.¹⁴Even so, the consistent finding that non-CG methylation appears to be confined to stem cell lines,^14,15 raises the possibility that cancer stem cells¹⁶ carry non-CG methylation while their nonstem progeny in the tumor carry only CG methylation. Given the expected paucity of cancer stem cells in a tumor cell population, it is unlikely that bisulfite sequencing would detect non-CG methylation in DNA isolated from tumor cells since the stem cell population is expected to be only a very minor component of tumor DNA. Published sequences obtained by bisulfite sequencing generally report only CG methylation, and to the best of our knowledge bisulfite sequenced tumor DNA specimens have not reported non-CG methylation. On the other hand, when sequences from cell lines have been reported, bisulfite-mediated genomic sequencing⁸ or ligation mediated PCR¹⁷ methylcytosine signals outside the CG site have been observed. In a more recent study plasmid DNAs carrying the Bcl2-major breakpoint cluster¹⁸ or human breast cancer DNA¹³ treated with bisulfite under non-denaturing conditions, cytosines outside the CG side were only partially converted on only one strand¹⁸ or at a symmetrical CWG site.¹³ In the breast cancer DNA study the apparent CWG methylation was not detected when the DNA was fully denatured before bisulfite treatment.¹³In both stem cell studies, non-CG methylation was attributed to the Dnmt3a,^14,15 a DNA methyltransferase with similarities to the plant DRM methyltransferase family¹⁹ and having the capacity to methylate non-CG sites when expressed in Drosophila melanogaster.¹⁵ DRM proteins however, possess a unique permuted domain structure found exclusively in plants¹⁹ and the associated RNA-directed non-CG DNA methylation has not been reproducibly observed in mammals despite considerable published^20–23 and unpublished efforts in that area. Moreover, reports where methylation was studied often infer methylation changes from 5AzaC reactivation studies²⁴ or find that CG methylation seen in plants but not non-CG methylation is detected.^21,22,25,26 In this regard, it is of interest that the level of non-CG methylation reported in stem cells corresponds to background non-CG methylation observed in vitro with human DNA methyltransferase I,²⁷ and is consistent with the recent report that cultured stem cells are epigenetically unstable.²⁸The function of non-CG methylation remains elusive. A role in gene expression has not been ruled out, as the studies above on Myf3 and B29 suggest.^9,10 However, transgene expression of the bacterial methyltransferase M.EcoRII in a human cell line (HK293), did not affect the CG methylation state at the APC and SerpinB5 genes²⁹ even though the promoters were symmetrically de novo methylated at ^mCWGs within each CCWGG sequence in each promoter. This demonstrated that CG and non-CG methylation are not mutually exclusive as had been suggested by earlier reports.^9,10 That observation is now extended to the human stem cell line methylome where CG and non-CG methylation co-exist.¹⁴ Gene expression at the APC locus was likewise unaffected by transgene expression of M.EcoRII. In those experiments genome wide methylation of the CCWGG site was detected by restriction analysis and bisulfite sequencing,²⁹ however stem cell characteristics were not studied.Many alternative functions can be envisioned for non-CG methylation, but the existing data now constrains them to functions that involve low levels of methylation that are primarily asymmetric. Moreover, inheritance of such methylation patterns requires low fidelity methylation. If methylation were maintained with high fidelity at particular CHG sites one would expect that the spontaneous deamination of 5-methylcytosine would diminish the number of such sites, so as to confine the remaining sites to those positions performing an essential function, as is seen in CG methylation.^30–33 However, depletion of CWG sites is not observed in the human genome.³⁴ Since CWG sites account for only about 50% of the non-CG methylation observed in the stem cell methylome¹⁴ where methylated non-CG sites carry only about 25% methylation, the probability of deamination would be about 13% of that for CWG sites that are subject to maintenance methylation in the germ line. Since mutational depletion of methylated cytosines has to have its primary effect on the germ line, if the maintenance of non-CG methylation were more accurate and more widespread, one would have had to argue that stem cells in the human germ lines lack CWG methylation. As it is the data suggests that whatever function non-CG methylation may have in stem cells, it does not involve accurate somatic inheritance in the germ line.The extensive detail on non-CG methylation in the H1 methylome¹⁴ raises interesting questions about the nature of this form of methylation in human cell lines. A key finding in this report is the contrast between the presence of non-CG methylation in the H1 stem cell line and its absence in the IMR90 human fetal lung fibroblast cell line.¹⁴ This suggests that it may have a role in the origin and maintenance of the pluripotent lineage.¹⁴By analogy with the well known methylated DNA binding proteins specific for CG methylation,³⁵ methylated DNA binding proteins that selectively bind sites of non-CG methylation are expected to exist in stem cells. Currently the only protein reported to have this binding specificity is human Dnmt1.^36–38 While Dnmt1 has been proposed to function stoichiometrically³⁹ and could serve a non-CG binding role in stem cells, this possibility and the possibility that other stem-cell specific non-CG binding proteins might exist remain to be been explored.Finally, the nature of the non-CG methylation patterns in human stem cell lines present potentially difficult technical problems in methylation analysis. First, based on the data in the H1 stem cell methylome,⁴⁰ a standard MS-qPCR for non-CG methylation would be impractical because non-CG sites are infrequent, rarely clustered and are generally characterized by partial asymmetric methylation. This means that a PCR primer that senses the 3 adjacent methylation sites usually recommended for MS-qPCR primer design^41,42 cannot be reliably found. For example in the region near Oct4 (Chr6:31,246,431), a potential MS-qPCR site exists with a suboptimal set of two adjacent CHG sites both methylated on the + strand at Chr6:31,252,225 and 31,252,237.^14,40 However these sites were methylated only in 13/45 and 30/52 reads. Thus the probability that they would both be methylated on the same strand is about 17%. Moreover, reverse primer locations containing non-CG methylation sites are generally too far away for practical bisulfite mediated PCR. Considering the losses associated with bisulfite mediated PCR⁴³ the likelihood that such an MS-qPCR system would detect non-CG methylation in the H1 cell line or stem cells present in a cancer stem cell niche^44,45 is very low.The second difficulty is that methods based on the specificity of MeCP2 and similar methylated DNA binding proteins for enriching methylated DNA (e.g., MIRA,⁴⁶ COMPARE-MS⁴⁷) will discard sequences containing non-CG methylation since they require cooperative binding afforded by runs of adjacent methylated CG sites for DNA capture. This latter property of the methylated cytosine capture techniques makes it also unlikely that methods based on 5-methylcytosine antibodies (e.g., meDIP⁴⁸) will capture non-CG methylation patterns accurately since the stem cell methylome shows that adjacent methylated non-CG sites are rare in comparison to methylated CG sites.¹⁴In summary, whether or not mammalian stem cells in general or human stem cells in particular possess functional plant-like methylation patterns is likely to continue to be an interesting and challenging question. At this point we can conclude that the non-CG patterns reported in human cells appear to differ significantly from the non-CG patterns seen in plants, suggesting that they do not have a common origin or function.     

Strigolactones' ability to regulate root development may be executed by induction of the ethylene pathway

The newly defined phytohormones strigolactones (SLs) were recently shown to act as regulators of root development. Their positive effect on root-hair (RH) elongation enabled examination of their cross talk with auxin and ethylene. Analysis of wild-type plants and hormone-signaling mutants combined with hormonal treatments suggested that SLs and ethylene regulate RH elongation via a common regulatory pathway, in which ethylene is epistatic to SLs. The SL and auxin hormonal pathways were suggested to converge for regulation of RH elongation; this convergence was suggested to be mediated via the ethylene pathway, and to include regulation of auxin transport.Key words: strigolactone, auxin, ethylene, root, root hair, lateral rootStrigolactones (SLs) are newly identified phytohormones that act as long-distance shoot-branching inhibitors (reviewed in ref. ¹). In Arabidopsis, SLs have been shown to be regulators of root development and architecture, by modulating primary root elongation and lateral root formation.^2,3 In addition, they were shown to have a positive effect on root-hair (RH) elongation.² All of these effects are mediated via the MAX2 F-box.^2,3In addition to SLs, two other plant hormones, auxin and ethylene, have been shown to affect root development, including lateral root formation and RH elongation.^4–6 Since all three phytohormones (SLs, auxin and ethylene) were shown to have a positive effect on RH elongation, we examined the epistatic relations between them by examining RH length.⁷ Our results led to the conclusion that SLs and ethylene are in the same pathway regulating RH elongation, where ethylene may be epistatic to SLs.⁷ Moreover, auxin signaling was shown to be needed to some extent for the RH response to SLs: the auxin-insensitive mutant tir1-1,⁸ was less sensitive to SLs than the wild type under low SL concentrations.⁷On the one hand, ethylene has been shown to induce the auxin response,^9–12 auxin synthesis in the root apex,^11,12 and acropetal and basipetal auxin transport in the root.^4,13 On the other, ethylene has been shown to be epistatic to SLs in the SL-induced RH-elongation response.⁷ Therefore, it might be that at least for RH elongation, SLs are in direct cross talk with ethylene, whereas the cross talk between SL and auxin pathways may converge through that of ethylene.⁷ The reduced response to SLs in tir1-1 may be derived from its reduced ethylene sensitivity;^7,14 this is in line with the notion of the ethylene pathway being a mediator in the cross talk between the SL and auxin pathways.The suggested ethylene-mediated convergence of auxin and SLs may be extended also to lateral root formation, and may involve regulation of auxin transport. In the root, SLs have been suggested to affect auxin efflux,^3,15 whereas ethylene has been shown to have a positive effect on auxin transport.^4,13 Hence, it might be that in the root, the SLs'' effect on auxin flux is mediated, at least in part, via the ethylene pathway. Ethylene''s ability to increase auxin transport in roots was associated with its negative effect on lateral root formation: ethylene was suggested to enhance polar IAA transport, leading to alterations in the quantity of auxin that unloads into the tissues to drive lateral root formation.⁴ Under conditions of sufficient phosphate, SL''s effect was similar to that of ethylene: SLs reduced the appearance of lateral roots; this was explained by their ability to change auxin flux.³ Taken together, one possibility is that the SLs'' ability to affect auxin flux and thereby lateral root formation in the roots is mediated by induction of ethylene synthesis.To conclude, root development may be regulated by a network of auxin, SL and ethylene cross talk.⁷ The possibility that similar networks exist elsewhere in the SLs'' regulation of plant development, including shoot architecture, cannot be excluded.