Arabidopsis thaliana and mouse. We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo. Furthermore, we show that simultaneous mutation of DDM1 and linker histone H1 in Arabidopsis reproduces the strong linker-specific methylation patterns of species that diverged from flowering plants and animals over a billion years ago. Our results indicate that in the absence of remodeling, nucleosomes are strong barriers to DNA methyltransferases. Linker-specific methylation can evolve simply by breaking the connection between nucleosome remodeling and DNA methylation.Cytosine methylation regulates essential genome functions across eukaryotes, but the fundamental question of whether nucleosomal or naked DNA is the preferred substrate of plant and animal methyltransferases remains unresolved. Here, we show that genetic inactivation of a single DDM1/Lsh family nucleosome remodeler biases methylation toward inter-nucleosomal linker DNA in  Living cells add chemical tags to their DNA to regulate which genes are switched on or off at any given time. These tags include methyl groups added to one of the letters of the DNA code called cytosine. Both plants and mammals need cytosine methylation to develop properly. This methylation also keeps sections of foreign DNA that may have invaded the cell in check.DNA inside the cell is tightly packed, wrapped around proteins to form spool-like structures called nucleosomes. Between each nucleosome is a short DNA segment called a linker region. The DNA wound into nucleosomes is generally inaccessible to other proteins, such as those that add methyl groups. Yet, in flowering plants and mammals, cytosine methylation occurs in both nucleosomes and in linker regions. It was not clear how DNA could be modified in the restrained setting of nucleosomes.Arabidopsis thaliana and mouse cells grown in the laboratory. In mutant plants lacking a nucleosome remodeler called DDM1, cytosine methylation occurred in the linker regions but not in the nucleosomes. Mouse cells lacking the mouse version of DDM1 also showed less cytosine methylation in the nucleosomes.Enzymes called nucleosome remodelers can loosen nucleosomes to allow other proteins to reach the DNA. Lyons and Zilberman asked whether cytosine methylation occurs on the nucleosome-bound DNA or if it requires enzymes like these to free the DNA from the constraints of the nucleosome. The experiments involved a plant called These results suggest that nucleosomes are barriers to the enzymes that modify DNA. Nucleosome remodeling enzymes like DDM1 can overcome these obstacles to enable cytosine methylation of nucleosome-wrapped DNA.These findings imply that cytosine methylation is more easily established and maintained on nucleosome-free DNA. Abnormal patterns of DNA methylation have been linked to medical conditions – such as neurological disorders and cancers – and to plant defects that hamper agriculture. A better understanding of the process may in the future lead to ways to correct problems with cytosine methylation in these different contexts. Cytosine methylation provides a mechanism to heritably alter the genome without permanent modification of the DNA sequence . In eukaAs implied by the above description of methylation pathways, DNA methylation represses TE transcription and transposition in plants and vertebrates . MethylaNucleosomes consist of about 147 bp of DNA tightly wrapped around an octameric complex of histone proteins, with the major and minor grooves of the double helix alternately facing toward and away from the histones roughly every 10 bp . NucleosArabidopsis plants with mutations in DDM1 suffer major methylation losses in all sequence contexts, primarily in heterochromatic TEs, though much smaller losses are also observed in genes , ddm1 mutants, compound h1ddm1 mutants, and in WT controls. We selected mononucleosomes in silico to avoid inconsistencies stemming from DNA electrophoretic mobility. Using biological replicates, we defined four groups of nucleosomes per genotype, from well-positioned to poorly-positioned , and anchored methylation analysis at the presumptive dyad (nucleosome center) -digested chromatin of  center) . We focu center)  and DDM1 center) .h1 mutant plants, and an overt depletion in ddm1 and h1ddm1 plants  computation  exhibits highly structured CG methylation that alternates with nucleosome positions  lack the winged-helix motif of canonical H1  were crossed to generate h1ddm1 and siblings for this study. Mutation of DDM1 causes permanent methylation losses at many loci that are not recovered after restoration of DDM1 activity . Following germination, they were transferred to soil and grown on a 16hr-light/8hr-dark long-day schedule. For all experiments described here, rosette leaves of 1 month old plants were used.ddm1-10 , h1.1 anand h1.2  Arabidopactivity . We therus study , was notArabidopsis thaliana rosette leaves with the DNeasy plant mini kit  per the manufacturer’s instructions. Libraries were prepared from roughly 500 ng of purified gDNA that was sheared to approximately 400 bp on a Misonix water bath sonicator, then purified using 1.2X volume of Agencourt Ampure beads . Following ligation of methylated Truseq sequencing adapters , bisulfite conversion of DNA was carried out according to manufacturer’s protocol  except without using carrier RNA. DNA was purified twice with 1.2X beads and converted a second time to ensure complete bisulfite conversion of unmethylated cytosine. NEB next indexing primers (cat. no. E7335S) were used for generating multiplexed libraries during PCR amplification of libraries.Genomic DNA (gDNA) was extracted from 1-month-old Leaves were flash frozen in liquid nitrogen, pulverized with mortar and pestle on dry ice, and the resulting material was subjected to vortexing in Trizol . Chloroform was then added at one-fifth the total volume and further vortexing was carried out until the solution appeared homogenous. RNA was subsequently pelleted in ice-cold isopropanol. The resuspended RNA was subjected to rRNA removal with Ribo-zero plant kit  according to the manufacturer’s protocol. 50 ng of ribo-depleted RNA was used for library preparation with the Scriptseq kit  following the manufacturer’s protocol but with the following modifications: the RNA fragmentation step was extended to 10 min, and the temperature was increased to 90°C.2, 60 mM KCl, 15 mM NaCl, 0.9% Triton X-100, 1 mM PMSF, 1X protease inhibitor cocktail) and allowed to thaw for 15 min. The suspension was then strained once through Miracloth  and the nuclear pellet was centrifuged in a swinging-bucket rotor at 2000 g in pre-chilled 5 ml conical tubes. Buffer was carefully aspirated from the loose pellet, which was subsequently resuspended in TM2  and washed twice in TM2. The pellet was then resuspended in pre-warmed MNase digestion buffer  and 25 units of MNase were added per 0.1 g initial starting material and incubated for 10 min at 37°C with periodic agitation; reactions were stopped with EGTA . This was followed by the addition of SDS to a final concentration of 0.625%, increasing the temperature to 75°C, addition of RNase A for 10 min, and the addition of proteinase-K for an additional 15 min. DNA was then extracted with phenol/chloroform, pelleted in 70% EtOH, then resuspended in TE pH 8.0.Approximately 1 g of pulverized flash frozen rosette leaf tissue from F4 generation plants was resuspended on ice in nuclei isolation buffer  using the Nugen Ovation kit (Cat. no. 0319) following the manufacturer’s protocol.All sequencing was carried out as single-end 100 bp reads, except MNase libraries, which were sequenced in paired-end 100 bp reads on Illumina HiSeq 2500 or −4000 at the QB3 Vincent Coates Genomic Sequencing Lab at UC Berkeley.http://dzlab.pmb.berkeley.edu/tools), with Bowtie settings allowing for up to two mismatches in the seed and reporting up to 10 matches for a given read. 100 bp paired-end MNase-seq reads were mapped using Bowtie2 . Shared nucleosomes  are defi‘Heterochromatic sequences’ in drm1drm2 or cmt2 were used to define DRM1/2 and CMT2 dependent DMRs, respectively. These windows also exhibited statistically significant decrease in mCHH from WT . We further filtered out DMRs that exhibited significant mCHH differences relative to WT in both genotypes, ensuring the DMRs are exclusive to the given genotype. After merging adjacent 50 bp DMR windows, there were 54,106 CMT2 DMRs and 8640 DRM1/2 DMRs. These coordinates are available in GEO accession GSE96994. All DMRs overlap at least one annotated TE.50 bp windows with at least 10% WT CHH methylation (mCHH) and with more than 30% loss of mCHH in h1 DMR plots are anchored to those DMRs which do not overlap any of the h1 group 1–4 nucleosomes).Methylation plots in http://www.ch.embnet.org/). The following sequences were used to perform multiple sequence alignment shown in Aureococcus anophagefferens, protein ID 72830, scaffold_256:8187–9341; Ostreococcus lucimarinus, protein ID 18993, Chr_20:191746–192084 (+); Emiliana huxleyi protein ID 448806/460675, scaffold_1716:1449–2181 (+)/scaffold_11:31908–32637 (-); Micromonas pusilla, protein ID 1714, scaffold_11:410445–411786, Arabidopsis thaliana H1.1 (AT1G06760), Neurospora crassa hH1 (ORF name NCU06863), and Mus musculus H1a (MGI:1931523).We aligned sequences with T-coffee  in normaCTCF sites  were useArabidopsis, 147 bp MNase-seq fragments were isolated from one of the biological replicates for each genotype. The resulting reads were used as anchors to calculate mean DNA methylation using bisulfite data . Perl scripts (http://dzlab.pmb.berkeley.edu/tools) were used to generate enrichment score matrices of mapped data around genomic features of interest. These matrices were imported to R . For instance, a 10 bp bin score of MNase-seq in sample A would be multiplied by 1 × 10ted to R  for furtted to R . Heatmapted to R  using thted to R . In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Detlef Weigel (Senior Editor) and three reviewers, one of whom, Richard Amasino (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Vincent Colot (Reviewer #2).Thank you for submitting this interesting study "DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision below to help you prepare a revised submission.In the consolidated review below, some parts are directly from the first round of review and a ** was added to those parts when a comment to address was within a paragraph.In this manuscript, Lyons and Zilberman examine the implication of the chromatin remodeler DDM1 and canonical histone H1 in the relationship between nucleosomes and DNA methylation in Arabidopsis. In previous work, the Zilberman lab showed that removal of canonical histone H1 leads to an increase in DNA methylation over heterochromatic TEs and a partial rescue of the well-documented loss of DNA methylation seen in ddm1. These observations lead to the conclusion that DDM1 provides DNA methyltransferases (MTases) with access to H1-containing heterochromatin .Numerous in vitro and in vivo studies using mammalian MTases and de novo DNA methylation assays have indeed established that the nucleosome core is an obstacle to DNA methylation. The authors should cite Felle et al., NAR 2011 as well as Baubec et al., Nature 2015 in their introductory paragraph . In addition, the authors should cite Takeshima et al., J. Mol. Biol. 2008; in this paper it was shown that histone H1 blocks DNA methylation of linker DNA in vitro.Lyons and Zilberman used MNase-seq and BS-seq to investigate directly DNA methylation in relation to nucleosome position in h1, ddm1 and h1ddm1 mutant plants as well as in WT controls. Key to their analysis is the definition of four groups of nucleosomes, from well-positioned to poorly-positioned. This distinction enabled the authors to report several new findings, which clarify the role of DDM1 in the maintenance of DNA methylation over H1-enriched nucleosomes.1) First, they show that heterochromatic nucleosomes, which are enriched in histone H1, have invariably high average CG methylation across the core and linker in WT controls. This is strong evidence that, in vivo, core and linker DNA are equally accessible to MET1, which is responsible for the maintenance of CG methylation. In marked contrast, average CHG and CHH methylation, which is much lower than average CG methylation, is preferentially located over linker DNA for these same nucleosomes. Thus, the MTases responsible for the perpetuation of this non-CG methylation across cell divisions preferentially access linker DNA in vivo.2/3) The second and key set of findings in this manuscript is that at presumably the same (**this is not clear from the text) well-positioned heterochromatic nucleosomes, the increase and decrease in average CG, CHG and CHH methylation observed in h1 and ddm1 mutant plants, is most and least pronounced over linker DNA, respectively. As could then be predicted, the partial rescue of methylation in the double mutant is also more pronounced over linker DNA. These findings are entirely consistent with the inhibitory effect in vitro of histone H1 on linker DNA methylation  and as noted above Takeshima et al. should be cited in this context. In addition, they suggest that DDM1 facilitates – to varying extents – the access of the different MTases responsible for CG, CHG and CHH methylation: not only to the core, but also to the linker, of H1-enriched, heterochromatic nucleosomes. **However, in the absence of genomic binding data for these MTases in WT and mutant backgrounds, indirect effects cannot be ruled out. This notwithstanding, the authors also used published nucleosome positioning and methylome data to show that loss of Lsh, the mammalian homolog of DDM1, causes a much greater depletion of DNA methylation over nucleosome cores than over linkers in mouse embryonic fibroblasts.4) Coming back to Arabidopsis, the authors then show that in contrast to TEs (which comprise most heterochromatic nucleosomes), genes have higher average CG methylation over nucleosome cores than over linker DNA in WT plants. This pattern is evident for exons but not for introns  and it remains largely unaltered in the three mutant backgrounds, except in the case of lowly expressed genes, perhaps because these genes undergo less transcription-coupled nucleosome displacement (it is not clear though if these genes are not part of TEs). The 10 bp methylation periodicity seen over nucleosome cores in all sequence contexts is also analyzed and results indicate that in the case of heterochromatic nucleosomes, it is dependent on DDM1. Based on this last result, the authors propose a model whereby DDM1 acts by releasing a loop of DNA from the surface of the histone octamer.In a last part, the authors show that a subset of heterochromatic nucleosomes that are well-positioned in the h1ddm1 double mutant exhibit the same DNA methylation pattern as seen in marine algae, namely a perfect alternation of low and high methylation over the nucleosome core and linker, respectively.In sum, this manuscript reports many new findings that are of broad interest. It adds nicely to the two other comprehensive studies by the Zilberman lab  and to that of Baubec et al. (Nature 2015) indicating that nucleosomes inhibit DNA methylation in vivo. In addition, it provides novel and compelling evidence that the linker histone H1 inhibits DNA methylation in vivo mainly by preventing access to linker DNA, consistent with in vitro data . Thus, as the authors suggest, the distinct patterns of DNA methylation observed among eukaryotes are likely determined in large part by whether or not a given species has well positioned nucleosomes and possesses the linker histone H1 and/or chromatin remodelers that can provide DNA MTases access to nucleosomes.5/6) Overall, the data as well as the analyses presented in the manuscript are solid and comprehensive. **However, most analyses are described too succinctly and it is often difficult to understand what exactly was done . Furthermore, the manuscript is difficult to read in places and I would recommend that the authors **subdivide it into three clearly identified sections  as well as several sub-sections in Results. This division will also help separate what is a fair interpretation of the findings reported in the manuscript, from the more speculative conclusions.7) One of the authors' main claims is that mutation of both DDM and Lsh1 bias methylation towards linker DNA. To support, and fully evaluate, this claim the following points should be addressed:a) The conclusion from b) For the lsh mutant, a meta-analysis is presented showing decreases in DNA methylation over well positioned nucleosomes and near WT levels of methylation in the linker regions; thus, methylation is skewed towards linkers. For ddm1/h1ddm1 mutants a similar trend is observed. However, more in-depth analyses should be presented to strengthen the assertion that LSH and DDM1 are acting similarly. For example, is Lsh activity also mainly associated with heterochromatin? Is the methylation periodicity in mammalian cells disrupted in lsh mutants?8) Another major point is that DDM1 enables the methylation of nucleosomal DNA by remodeling the chromatin such that DNA methylation actually occurs on a DNA loop and not directly on nucleosome bound DNA.Here the authors suppose (without any data) that this loop would still retain a bias in DNA methylation resulting in a periodic pattern matching previous in vivo findings. To test their model, the periodicity of DNA methylation was then assessed in a ddm1 mutant background. However, as this mutant causes global decreases in DNA methylation, disruption of the periodic behavior is hard to assess. Furthermore, the decrease in periodicity could either be because the DNA loops aren't created so methylation is blocked or because DDM1 serves a different function that allows MET1 to directly methylate nucleosomal DNA. Thus, these in vivo experiments remain inconclusive. To support their conclusions, in vitro remodeling assays showing that DDM1 activity creates accessible DNA loops that can be methylated by MET1 would be highly informative, although such assays are clearly not trivial. In the absence of such assays, the authors should note the alternative interpretations noted above.9) A final point of the paper is that the decoupling of chromatin remodeling and DNA methylation could explain some of the differences in global methylation patterns observed between species.Here the authors find that a subset of well positioned, heterochromatin-localized nucleosomes (cluster C2) have strong linker methylation and low nucleosome methylation in the h1ddm1 background. Based on the similarity of this DNA methylation pattern with those observed in algae, it is proposed that decoupling methylation and remodeling is a mechanism employed to generate such patterns in organisms with well positioned nucleosomes. However, this model is not further vetted. In this case, a phylogenetic analysis correlating the presence or absence of DDM1 or H1 orthologs with linker-specific methylation patterns might provide additional support for the authors claims.Although in general we appreciate brevity, some elaboration here would be useful to the reader. Do you think this phenomenon is a result of genetic drift in which in some organisms the loss of one component is not selected against or might it provide some selective advantage? In this section is the sentence: "These species are interspersed on the tree of life with species that do not limit methylation to linkers ." FigureSpecific comments:1) Main text, first paragraph: Replace Jones and Baylin, 2007 with Baylin and Jones, CSH Persp. Biol. 2016, which is more appropriate and recent.2) Main text, first paragraph: To be consistent with the first part of the sentence, I would be more general here and provide a review instead of Ong-Abdullah et al., 2015.3) Main text, fourth paragraph: It is not clear if the four groups of nucleosomes were determined for each of the four genotypes or solely for the WT and then maintained for the analysis of the three mutant genotypes? I suspect that it is the latter, but the description in Materials and methods suggests otherwise. How can meaningful comparisons be made if the four groups of nucleosomes differ for each genotype?4) The authors could easily complement their analysis of heterochromatic nucleosomes by considering separately those that have CMT2-dependent CHH methylation and those that are targeted by DRM1/2 instead. This additional analysis could also prove very informative.5) Main text, fourth paragraph: Make also reference to the Materials and methods section ("Description of Arabidopsis genome features").6) Main text, seventh paragraph: “7) Main text, ninth paragraph: Are all genes non TE-genes? And what about TEs? Please provide about how the TE class was defined and how it differs from the heterochromatic nucleosomes analyzed in the first part.8) Main text, tenth paragraph: The fact that gene methylation is enriched over nucleosomes and that DDM1 is not required for this type of methylation except in the case of lowly expressed genes suggests that other chromatin remodelers are involved. However, at this stage this is only a hypothesis and the authors should therefore tone down the last sentence of this paragraph.9) Materials and methods:- Why was the mutant allele ddm1-10 chosen over the much more commonly studied allele ddm1-2, used for instance in Zemach et al., 2013.- Which generation was used for ddm1 and h1ddm1? This is important to know as the demethylation of heterochromatic sequences increases with the number of generations.- Were DNA, RNA and nucleosomes extracted from the same starting biological material?10) 11) The legend of In 12) It is not clear from the methods if the same stage leaf tissue was used for the plant DNA methylation and nucleosome profiling experiments. As these features can vary during development, this point should be clarified.Thank you for resubmitting your work entitled "DDM1 and Lsh remodelers allow methylation of DNA wrapped in nucleosomes" for further consideration at eLife. Your revised article has been favorably evaluated by Detlef Weigel (Senior Editor), a Reviewing Editor, and two reviewers.There are a few minor points to address that the reviewers and I think would make your paper even more clear to the eLife readership.1)We suggest a more direct formulation. "DNA methylation at DRM1/2-dependent loci is actually enriched around well-positioned nucleosomes in WT . This en2) Discussion, fourth paragraph, last sentence: This sentence is ambiguous. Gene body methylation is affected by MET1, with no contribution from the other methyltransferases. The ddm1 mutation affects only a small subset of body methylated genes.3) Re the statement in the Abstract "We find that DDM1 enables methylation of DNA bound to the nucleosome, thus demonstrating that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo." The first half of the statement is well supported, but the data do not demonstrate that nucleosome-free DNA is the preferred substrate. Rather, the data support this hypothesis. It is up to you how to re-phrase, but an example of re-phrasing would be "We find that DDM1 enables methylation of DNA bound to the nucleosome, consistent with a model in which nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo."4) In several cases, you draw specifically on the effects of h1 on CG methylation (see quoted text below). However, the effects of h1 on CG methylation are minimal. Indeed, when plotted relative to WT levels in "CG methylation is relatively evenly distributed with respect to nucleosomes in WT plants, and shows a weak nucleosomal depletion in h1 mutants .""Without H1, linker DNA becomes even more accessible, producing the observed slight core-to-linker methylation differential in h1 mutants .”5) The text now makes clear that the methylation data shown in 6) The implications of the following statement are unclear."However, this appearance is almost certainly caused by the small size of DRM1/2-dependent TEs, because an analysis anchored to the centers of differentially methylated regions  between drm1drm2 mutants and wild-type that occur in regions with very poorly positioned nucleosomes (those outside the four groups defined in can cause gene silencing[…]"7) To avoid confusion the word "can" should be added to this sentence of the Introduction: "Methylation also regulates endogenous genes: methylation close to the transcriptional start site  In the consolidated review below, some parts are directly from the first round of review and a ** was added to those parts when a comment to address was within a paragraph.In this manuscript, Lyons and Zilberman examine the implication of the chromatin remodeler DDM1 and canonical histone H1 in the relationship between nucleosomes and DNA methylation in Arabidopsis. In previous work, the Zilberman lab showed that removal of canonical histone H1 leads to an increase in DNA methylation over heterochromatic TEs and a partial rescue of the well-documented loss of DNA methylation seen in ddm1. These observations lead to the conclusion that DDM1 provides DNA methyltransferases (MTases) with access to H1-containing heterochromatin .Numerous in vitro and in vivo studies using mammalian MTases and de novo DNA methylation assays have indeed established that the nucleosome core is an obstacle to DNA methylation. The authors should cite Felle et al., NAR 2011 as well as Baubec et al., Nature 2015 in their introductory paragraph . In addition, the authors should cite Takeshima et al., J. Mol. Biol. 2008; in this paper it was shown that histone H1 blocks DNA methylation of linker DNA in vitro.We have added the following sentence to the Introduction section:“However, mammalian methyltransferase activity is inhibited by nucleosomes and H1 in vitro , and regions of the human and mouse genomes with reliably positioned nucleosome arrays organized by CTCF binding exhibit preferential methylation of linker DNA .”We are reluctant to cite Felle et al. in support of linker-biased methylation in vivo because the in vivo experiments in this paper are deeply flawed. The authors find that larger MNase-digested fragments, which represent multiple nucleosomes and are enriched for linker DNA, are more methylated than shorter mononucleosome fragments. They infer that linkers are more methylated than nucleosomes, but this assumes all genomic sequences are equally accessible to MNase. Instead, the shorter fragments come from more accessible and less methylated euchromatin, explaining the enrichment of methylation on longer fragments.Lyons and Zilberman used MNase-seq and BS-seq to investigate directly DNA methylation in relation to nucleosome position in h1, ddm1 and h1ddm1 mutant plants as well as in WT controls. Key to their analysis is the definition of four groups of nucleosomes, from well-positioned to poorly-positioned. This distinction enabled the authors to report several new findings, which clarify the role of DDM1 in the maintenance of DNA methylation over H1-enriched nucleosomes.1) First, they show that heterochromatic nucleosomes, which are enriched in histone H1, have invariably high average CG methylation across the core and linker in WT controls. This is strong evidence that, in vivo, core and linker DNA are equally accessible to MET1, which is responsible for the maintenance of CG methylation. In marked contrast, average CHG and CHH methylation, which is much lower than average CG methylation, is preferentially located over linker DNA for these same nucleosomes. Thus, the MTases responsible for the perpetuation of this non-CG methylation across cell divisions preferentially access linker DNA in vivo.2/3) The second and key set of findings in this manuscript is that at presumably the same (**this is not clear from the text) well-positioned heterochromatic nucleosomes, the increase and decrease in average CG, CHG and CHH methylation observed in h1 and ddm1 mutant plants, is most and least pronounced over linker DNA, respectively. As could then be predicted, the partial rescue of methylation in the double mutant is also more pronounced over linker DNA.We have clarified the differences between nucleosome sets and added a detailed explanation regarding why we used nucleosomes defined separately for each genotype in These findings are entirely consistent with the inhibitory effect in vitro of histone H1 on linker DNA methylation  and as noted above Takeshima et al. should be cited in this context.We have added a citation to Takeshima et al. (2008).In addition, they suggest that DDM1 facilitates – to varying extents – the access of the different MTases responsible for CG, CHG and CHH methylation: not only to the core, but also to the linker, of H1-enriched, heterochromatic nucleosomes. **However, in the absence of genomic binding data for these MTases in WT and mutant backgrounds, indirect effects cannot be ruled out.We have added the following comment to the beginning of our Discussion section:“It remains formally possible that DDM1/Lsh remodelers permit methyltransferases to work directly on the nucleosome surface or promote catalysis without affecting methyltransferase access.”This notwithstanding, the authors also used published nucleosome positioning and methylome data to show that loss of Lsh, the mammalian homolog of DDM1, causes a much greater depletion of DNA methylation over nucleosome cores than over linkers in mouse embryonic fibroblasts.4) Coming back to Arabidopsis, the authors then show that in contrast to TEs (which comprise most heterochromatic nucleosomes), genes have higher average CG methylation over nucleosome cores than over linker DNA in WT plants. This pattern is evident for exons but not for introns  and it remains largely unaltered in the three mutant backgrounds, except in the case of lowly expressed genes, perhaps because these genes undergo less transcription-coupled nucleosome displacement (it is not clear though if these genes are not part of TEs).We have added a paragraph to the Discussion regarding gene body methylation and exon vs. intron DNA methylation.The 10 bp methylation periodicity seen over nucleosome cores in all sequence contexts is also analyzed and results indicate that in the case of heterochromatic nucleosomes, it is dependent on DDM1. Based on this last result, the authors propose a model whereby DDM1 acts by releasing a loop of DNA from the surface of the histone octamer.In a last part, the authors show that a subset of heterochromatic nucleosomes that are well-positioned in the h1ddm1 double mutant exhibit the same DNA methylation pattern as seen in marine algae, namely a perfect alternation of low and high methylation over the nucleosome core and linker, respectively.In sum, this manuscript reports many new findings that are of broad interest. It adds nicely to the two other comprehensive studies by the Zilberman lab  and to that of Baubec et al. (Nature 2015) indicating that nucleosomes inhibit DNA methylation in vivo. In addition, it provides novel and compelling evidence that the linker histone H1 inhibits DNA methylation in vivo mainly by preventing access to linker DNA, consistent with in vitro data . Thus, as the authors suggest, the distinct patterns of DNA methylation observed among eukaryotes are likely determined in large part by whether or not a given species has well positioned nucleosomes and possesses the linker histone H1 and/or chromatin remodelers that can provide DNA MTases access to nucleosomes.5/6) Overall, the data as well as the analyses presented in the manuscript are solid and comprehensive. **However, most analyses are described too succinctly and it is often difficult to understand what exactly was done . Furthermore, the manuscript is difficult to read in places and I would recommend that the authors **subdivide it into three clearly identified sections  as well as several sub-sections in Results. This division will also help separate what is a fair interpretation of the findings reported in the manuscript, from the more speculative conclusions.We have reorganized the paper into separate Introduction, Results, and Discussion sections. We also added Results subsections and moved more speculative elements of our interpretation of data to the Discussion. We have expanded the explanations of our methods as well as the results, and much of the interpretation of the results has been expanded in the Discussion.7) One of the authors' main claims is that mutation of both DDM and Lsh1 bias methylation towards linker DNA. To support, and fully evaluate, this claim the following points should be addressed:a) The conclusion from We have added supplemental figures and explanatory text in the results to help clarify this issue for Arabidopsis data. We define groups of positioned nucleosomes separately for each genotype in We have now added supplemental data to illustrate that our conclusions do not change when analyzing sets of well-positioned nucleosomes shared by different genotypes . This fiRegarding nucleosome positioning in Lsh null cells, we unfortunately do not have Lsh null nucleosome data. We feel that CTCF sites are appropriate for anchoring analysis of methylation in this context; in essence, these can be thought of as ‘shared well-positioned nucleosomes’. Nucleosome positioning at CTCF sites in humans and mouse has been well-described and is generally invariant when averaged across a population of cells  as we do in the current work. Furthermore, this subset of CTCF sites is distant from potentially disruptive positioning cues such as gene promoters .Indeed, the CTCF sites we used here are well bound by CTCF regardless of levels of DNA methylation  and following the addition of DNMT3a to mutant cells, mCG appears in the presumptive linker DNA of these regions . The observation that average DNA methylation in Lsh null cells peaks in the presumptive linker regions at near-WT levels provides strong evidence that the nucleosomes are positioned as in WT. There is also precedent for our analysis: Baubec et al. (2015) show MNase-seq from WT cells  in relation to methylation data from Dnmt-triple KO mutants.b) For the lsh mutant, a meta-analysis is presented showing decreases in DNA methylation over well positioned nucleosomes and near WT levels of methylation in the linker regions; thus, methylation is skewed towards linkers. For ddm1/h1ddm1 mutants a similar trend is observed. However, more in-depth analyses should be presented to strengthen the assertion that LSH and DDM1 are acting similarly. For example, is Lsh activity also mainly associated with heterochromatin?We have added the following sentence to the Introduction section:“Knockout of mouse Lsh likewise causes major depletion of DNA methylation from repetitive heterochromatin , with more variable effects on methylation in genes and other sequences .”Is the methylation periodicity in mammalian cells disrupted in lsh mutants?We have added an analysis of Lsh-/- mCG periodicity to 8) Another major point is that DDM1 enables the methylation of nucleosomal DNA by remodeling the chromatin such that DNA methylation actually occurs on a DNA loop and not directly on nucleosome bound DNA.Here the authors suppose (without any data) that this loop would still retain a bias in DNA methylation resulting in a periodic pattern matching previous in vivo findings. To test their model, the periodicity of DNA methylation was then assessed in a ddm1 mutant background. However, as this mutant causes global decreases in DNA methylation, disruption of the periodic behavior is hard to assess. Furthermore, the decrease in periodicity could either be because the DNA loops aren't created so methylation is blocked or because DDM1 serves a different function that allows MET1 to directly methylate nucleosomal DNA. Thus, these in vivo experiments remain inconclusive. To support their conclusions, in vitro remodeling assays showing that DDM1 activity creates accessible DNA loops that can be methylated by MET1 would be highly informative, although such assays are clearly not trivial. In the absence of such assays, the authors should note the alternative interpretations noted above.We have separated the model now  and the 9) A final point of the paper is that the decoupling of chromatin remodeling and DNA methylation could explain some of the differences in global methylation patterns observed between species.Here the authors find that a subset of well positioned, heterochromatin-localized nucleosomes (cluster C2) have strong linker methylation and low nucleosome methylation in the h1ddm1 background. Based on the similarity of this DNA methylation pattern with those observed in algae, it is proposed that decoupling methylation and remodeling is a mechanism employed to generate such patterns in organisms with well positioned nucleosomes. However, this model is not further vetted. In this case, a phylogenetic analysis correlating the presence or absence of DDM1 or H1 orthologs with linker-specific methylation patterns might provide additional support for the authors claims.We have added a phylogenetic analysis of histone H1 to illustrate that, as the reviewer suggested, distribution of canonical linker histone may be informative . Indeed,Although in general we appreciate brevity, some elaboration here would be useful to the reader. Do you think this phenomenon is a result of genetic drift in which in some organisms the loss of one component is not selected against or might it provide some selective advantage?We suspect that the loss of H1 and gain of linker specific methylation may confer a selective advantage. We have added the following passage to the Discussion:“The linker-specific methylation patterns almost certainly offer a selective advantage to the species that have them, in part by contributing to nucleosome positioning . Thus, loss of H1 could be advantageous in the presence of a linker-specific methylation system, and reliable nucleosome positioning a consequence of such a system.”In this section is the sentence: "These species are interspersed on the tree of life with species that do not limit methylation to linkers ." FigureThis tree may have been improperly referenced in the original submission. Specific comments:1) Main text, first paragraph: Replace Jones and Baylin, 2007 with Baylin and Jones, CSH Persp. Biol. 2016, which is more appropriate and recent.We switched this reference as suggested.2) Main text, first paragraph: To be consistent with the first part of the sentence, I would be more general here and provide a review instead of Ong-Abdullah et al., 2015.We have added a 2017 review paper by Springer and Schmitz.3) Main text, fourth paragraph: It is not clear if the four groups of nucleosomes were determined for each of the four genotypes or solely for the WT and then maintained for the analysis of the three mutant genotypes? I suspect that it is the latter, but the description in Materials and methods suggests otherwise. How can meaningful comparisons be made if the four groups of nucleosomes differ for each genotype?Please see general comment 7a and response above.4) The authors could easily complement their analysis of heterochromatic nucleosomes by considering separately those that have CMT2-dependent CHH methylation and those that are targeted by DRM1/2 instead. This additional analysis could also prove very informative.An analysis of differentially-methylated CHH regions has been added as a new main figure, 5) Main text, fourth paragraph: Make also reference to the Materials and methods section ("Description of Arabidopsis genome features").We have added a reference to Arabidopsis genome features .6) Main text, seventh paragraph: “This has been corrected.7) Main text, ninth paragraph: Are all genes non TE-genes? And what about TEs? Please provide about how the TE class was defined and how it differs from the heterochromatic nucleosomes analyzed in the first part.Clarifying notes have been added to the Results and Materials and methods where appropriate. In particular, our gene annotations exclude TEs.8) Main text, tenth paragraph: The fact that gene methylation is enriched over nucleosomes and that DDM1 is not required for this type of methylation except in the case of lowly expressed genes suggests that other chromatin remodelers are involved. However, at this stage this is only a hypothesis and the authors should therefore tone down the last sentence of this paragraph.We have edited this claim:“Our results demonstrate that DDM1 facilitates methylation of nucleosome-wrapped DNA in a subset of genes as well as in heterochromatin.”We also now describe the possibility of other remodelers facilitating genic methylation in the discussion, to make clear that this is a hypothesis.9) Materials and methods:- Why was the mutant allele ddm1-10 chosen over the much more commonly studied allele ddm1-2, used for instance in Zemach et al., 2013.To prevent complications of using inbred ddm1 alleles that have existed as homozygous mutants and retain methylation defects even when backcrossed to WT, we used this allele, which we obtained in a heterozygous state. We have added an explanation to the Materials and methods section to convey this.- Which generation was used for ddm1 and h1ddm1? This is important to know as the demethylation of heterochromatic sequences increases with the number of generations.F4. The reason for is that – as explained in forthcoming work related to this manuscript – we have found h1ddm1 CG methylation at TEs decreases from F2 to F3, whereas by the F4 methylation is stable for the most part.- Were DNA, RNA and nucleosomes extracted from the same starting biological material?DNA for bisulfite sequencing was taken from the same tissue type and the same generation as the MNase samples (F4), but not from exactly the same biological material. RNA was from the WT F3 from the parental h1 x ddm1/+ cross. The RNA-seq data were used only to define gene expression deciles.10) We have added CHH methylation analysis to the main figure.I would also recommend for each of the three sequence contexts merging the four graphs, so as to make more apparent the gains and losses of methylation. This is particularly important for CHH methylation, which is low to start with compared to CHG and especially CG methylation and which show modest fluctuations in absolute values. Indicating levels relative to WT could improve visualization further.We agree that showing methylation around nucleosomes relative to WT helps to convey the phenotype more clearly. We have added a panel, 11) The legend of We have added heatmaps of methylation for the non-CG contexts to the supplement ; they shIn This is now 12) It is not clear from the methods if the same stage leaf tissue was used for the plant DNA methylation and nucleosome profiling experiments. As these features can vary during development, this point should be clarified.We have added a sentence at the end of the first Materials and methods paragraph to clarify this point. For all experiments, rosette leaves of 1-month old plants were used.There are a few minor points to address that the reviewers and I think would make your paper even more clear to the eLife readership.1)We suggest a more direct formulation. "DNA methylation at DRM1/2-dependent loci is actually enriched around well-positioned nucleosomes in WT . This enThe issue here is similar to the point above – we did not present our data clearly and described the results in an overly conservative and confusing way. CG and CHG methylation is actually depleted over nucleosomes very similarly at DRM- and CMT2-dependent loci, which shouldn’t be surprising given that the same methyltransferases  are responsible. CHH methylation is also depleted over nucleosomes at DRM-dependent loci, but mutating H1 or DDM1 doesn’t make much difference. We now illustrate this by plotting methylation in Arabidopsis TEs can be roughly grouped into heterochromatic and euchromatic elements . […] Due to the strong linker enrichment of methylation in h1ddm1 heterochromatin  Discussion, fourth paragraph, last sentence: This sentence is ambiguous. Gene body methylation is affected by MET1, with no contribution from the other methyltransferases. The ddm1 mutation affects only a small subset of body methylated genes.We altered this sentence to:ddm1 mutants  Re the statement in the Abstract "We find that DDM1 enables methylation of DNA bound to the nucleosome, thus demonstrating that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo." The first half of the statement is well supported, but the data do not demonstrate that nucleosome-free DNA is the preferred substrate. Rather, the data support this hypothesis. It is up to you how to re-phrase, but an example of re-phrasing would be "We find that DDM1 enables methylation of DNA bound to the nucleosome, consistent with a model in which nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo."We changed this sentence of the Abstract to:“We find that DDM1 enables methylation of DNA bound to the nucleosome, suggesting that nucleosome-free DNA is the preferred substrate of eukaryotic methyltransferases in vivo.”4) In several cases, you draw specifically on the effects of h1 on CG methylation (see quoted text below). However, the effects of h1 on CG methylation are minimal. Indeed, when plotted relative to WT levels in "CG methylation is relatively evenly distributed with respect to nucleosomes in WT plants, and shows a weak nucleosomal depletion in h1 mutants .""Without H1, linker DNA becomes even more accessible, producing the observed slight core-to-linker methylation differential in h1 mutants .”We thank the reviewers for pointing out that our data presentation in To present our data clearly and accurately, we changed h1 mutant plants, and an overt depletion in ddm1 and h1ddm1 plants (h1ddm1 plants (“CG methylation shows a mild but clear depletion over well-positioned nucleosomes in WT and 1 plants .”5) The text now makes clear that the methylation data shown in We added the relevant nucleosome numbers to the legend of “Nucleosome numbers: 19122 for WT, 4267 shared by WT and h1ddm1 (22.3% of WT), 22745 for h1ddm1, 6364 shared by h1 and h1ddm1 (28.0% of h1ddm1).”6) The implications of the following statement are unclear."However, this appearance is almost certainly caused by the small size of DRM1/2-dependent TEs, because an analysis anchored to the centers of differentially methylated regions  between drm1drm2 mutants and wild-type that occur in regions with very poorly positioned nucleosomes (those outside the four groups defined in Please see our response to comment 1 above.7) To avoid confusion the word "can" should be added to this sentence of the Introduction: "Methylation also regulates endogenous genes: methylation close to the transcriptional start site can cause gene silencing[…]"We made the suggested change.


The NHS Bowel Cancer Screening Programme (BCSP) in England does not involve general practitioners (GPs). Uptake is ∼58%. The Practice Endorsed Additional Reminder Letter (PEARL) study piloted a GP-endorsed reminder letter.General practices in Wessex with uptake <55%  were invited to participate. Subjects who had been invited for screening, sent a standard 28-day BCSP reminder letter but had not returned a test kit within 30 days of the standard reminder were sent a second reminder letter bearing the GP’s letterhead and signature. Uptake was compared between PEARL and non-PEARL practices by standardised uptake ratio . In addition, 25 non-PEARL practices were matched with PEARL practices for prior prevalent uptake and number of invitees.P<0.001) for all invitees and 2.18  for invitees who had not returned a kit following the standard reminder.Twenty-five practices agreed to participate. A total of 3149 GP-endorsed reminders were sent. Uptake in the PEARL practices was 54% compared with 51% in the matched-control practices. The adjusted RR for uptake was 1.08 (95% CI: 1.05, 1.11, The GP-endorsed reminder was associated with significantly increased uptake among subjects not responding to the standard reminder letter. Worldwide, colorectal cancer (CRC) is a major health burden; in 2012, nearly 1.4 million cases were diagnosed and there were 694 000 deaths  test result are referred for a follow-up investigation; if the test is negative , participants will be invited to be screened again in 2 years’ time until they reach the age of 75 . A reminder letter is sent to subjects where no action has occurred on the Bowel Cancer Screening System within 28 days of being sent a test kit. Screening episodes are closed if there is no response 17 weeks after the kit was sent.In England, ‘uptake’ of screening is defined as the proportion of invited subjects that is adequately screened (definitive positive or definitive negative test result) within 26 weeks of invitation. Overall uptake in England was 58.2% in 2014/2015 , considerably lower than in the breast and cervical screening programmes  project was to develop and pilot a sustainable intervention to engage subjects in bowel cancer screening. The project was designed to test whether an additional reminder letter on GP-headed paper and with the signature of the subject’s GP could encourage uptake. It is the result of collaboration between the BCSP Southern Hub, a group of GPs in Wessex  working for Macmillan Cancer Support and Cancer Research UK, in partnership with the Wessex Strategic Clinical Network.The BCSP Southern Hub serves the south of England (excluding London), sending 20 000 invitations for screening every week. The PEARL project tested the intervention in the target area of Wessex. All general practices in Wessex with an uptake of less than 55%  were invited to participate. These practices were typically in areas of socioeconomic deprivation. The PEARL intervention targeted all subjects aged 60–74 years (inclusive), registered with a participating practice who were invited to take part in screening and had not responded to the standard 28-day BCSP reminder letter.Eligible practices were contacted by e-mail and regular mail to invite them to participate. If no reply was received, they were contacted by telephone. The deadline for agreement to participate was 31 May 2014. Participating practices agreed to receive an educational 1-h visit to learn about the BCSP, the PEARL project and to discuss the practice-specific statistical cancer profile—the National Cancer Intelligence Network general practice profile. Practices were offered a financial incentive (£250) for participating in the project.Each practice signed a consent form and a confidentiality agreement and data exchange form, which set out the responsibilities of both the Hub and the GP practice .The text of a standard second reminder letter  was apprEach participating practice sent the Hub a paper or scanned electronic copy of the practice letterhead and a digital GP signature. One GP signed the letter for all patients from the practice. An electronic letter template was constructed for each practice.The PEARL intervention took place in Wessex between September 2014 and October 2015 and was designed to run for a fixed period of time. The flowchart in GPs returned completed spreadsheets to the Hub by secure e-mail.Ten working days after the initial list was sent, the list of subjects on returned GP spreadsheets was checked against a new download of subject-level data from Bowel Cancer Screening System to ensure that subjects remained non-respondent, were still registered with the same GP, and that there was no record of death. GP-endorsed reminder letters were printed for all eligible subjects with the appropriate GP letterhead and signature. This process was repeated 15 working days after the initial list for any GP spreadsheets returned late.In any 1 month, if no non-responders were identified for a participating practice, the practice was notified by e-mail that they would not receive a list that month. Practices that did not return a completed spreadsheet were sent a reminder e-mail 5 working days after the spreadsheet was sent.The average date of sending a GP-endorsed reminder was 99 days after initial invitation. Subjects in PEARL-registered practices not sent a reminder and in the non-PEARL practices were assigned an index date, the date when a PEARL reminder would have been sent if the practices were participating and the subject had not yet returned an adequate kit. Uptake, the primary outcome measure, was defined as the proportion of subjects  that was adequately screened. Uptake was compared between PEARL practices and all other practices in the Southern Hub region with a prevalent uptake of less than 55%. The comparison group included all routine screened Southern Hub subjects with an invitation on days that PEARL subjects were invited.Subject variables were sex, age-group, and screening history . In addition, a measure of social deprivation (index of multiple deprivation (IMD)) was assigned for each subject with reference to the English indices of deprivation. The IMD scores were grouped into quintiles based on national distributions with the use of predefined national cutoffs.Both the 25 PEARL-registered practices and the 1575 comparison practices were specified to have prevalent screen participation rates of less than 55% prior to the intervention. However, even within this range, the comparison practices had substantially higher prior participation rates than the PEARL-registered practices (see Results section). We therefore analysed the data analogously to standardised incidence or mortality ratios. Using logistic regression, in subjects within the comparison practices only, we estimated the effects of age, sex, prior prevalent participation, IMD quintile, episode type , and episode sequence on the probability of participation. These were applied to the subjects in the PEARL-registered practices to calculate the expected proportion of participants in these practices , and IMD quintile.As a check on our method, we also selected 25 non-PEARL practices, matched to the PEARL-registered practices by previous prevalent participation rates and number of invitees. We repeated our primary analysis, directly comparing participation rates in PEARL-registered practices with those in the matched non-PEARL practices. Analysis for this comparison was performed using inverse variance weighted average estimation conditioning on matched set . All anaThe PEARL project protocol was approved by the NHS BCSP Research Committee in March 2015 (reference ID 148). The Office for Data Release granted permission for sharing BCSP data with Professor Stephen Duffy (statistician) in November 2015 (reference ODR1516_154). Patient consent was not required under Section 251 of the 2006 NHS Act. The PEARL project is part of the ACE  programme, NHS England’s initiative supported by Cancer Research UK and Macmillan Cancer Support to diagnose cancer early and improve outcomes. Funding was received from the National Cancer Intelligence Network.n=1–7) that received no list. The mean number of subjects on each list was 16.02 (range 1–103).One hundred twenty-four (38.3%) lists were not returned. Two of the practices that signed up to the PEARL intervention did not return any of the lists.Of the 43 eligible practices approached, 25 agreed to participate in the PEARL study. Over the 14-month study period, 324 non-participating subject lists were sent to GP practices. Seventeen practices had lists to review every month. For 11 of the study months, there were practices (n=10)Patients receiving end-of-life care (n=20)Already having regular colonoscopy surveillance (n=4)Recent (<12 months) diagnosis of bowel cancer (n=5)Unable to have colonoscopy (n=122).Other (One hundred sixty-one (5.11%) subjects were deemed unsuitable by the GP to receive a reminder letter for the following reason:There were 12 878 invitees in the PEARL-registered practices and 1 248 689 in the non-PEARL-registered practices. A total of 3149 GP-endorsed reminder letters were sent.P<0.001) difference between the participation rates in the PEARL practices and that expected from the non-PEARL, with 6914 (54%) participation vs 6543 (51%) expected. In those who qualified for a reminder, that is, those who had not returned a completed kit by the index date, there was also a highly significant difference (P<0.001) with 362 (7%) participation in the PEARL practices compared with 167 (3%) expected. The direct comparison of the 25 PEARL practices with the 25 matched practices showed slightly stronger but essentially similar results ( results .P=0.001). No other significant heterogeneity of the PEARL intervention effect between subgroups was observed.In the corresponding results in the lower half of the table, for those with no return of an adequate kit before the index date, all subgroups showed highly significant results, with an approximate doubling of the rate of participation, typically from 2–3 to 6–8%. Again, there was significant heterogeneity of the effect by age, with a stronger effect of the PEARL intervention in those older than 65 years. No other significant heterogeneity of the intervention effect was observed between subgroups.et al , in general practices with participation rates below the national average. There is already evidence that primary care endorsement improves participation in screening for a number of cancers   is likely to have had a positive effect on the subject’s attitude. Other research has demonstrated that uptake of screening can be improved by direct contact in person with a GP or other health professional  and the link between deprivation and low uptake of bowel cancer screening has been firmly established. It may be that the observations should not be extrapolated to all GP surgeries on a national scale.A key consideration for the study was the GP–patient relationship if a letter was coming on GP letter headed paper. This was the reason the GP was asked to review a list of their patients who had not responded and who were on the list to receive a reminder. It is notable that a reasonable number (5.11%) of subjects were deemed unsuitable to receive the letter from the GP suggesting that sending out GP-personalised letters without having a GP review in advance might be inappropriate.vs 57.5%). The authors concluded that, given the willingness shown by GPs to endorse the BCSP, the small one-off cost incurred to modify the standard invitation letter and the overall increase in uptake, the BCSP should consider adding the GP endorsement to the screening invitation letter. The London BCSP Hub have implemented the GP endorsement.As part of the ASCEND randomised controlled trial in England , investigators sought permission from general practices to allow the practice name to be added to the standard BCSP pre-invitation letter (Compared with the ASCEND GP-endorsed pre-invitation intervention (We have developed a robust and sustainable method to send GP-endorsed letters to non-responders. A GP-endorsed second reminder letter significantly increased uptake, by about 3 percentage points, both as a proportion of all invitees or only those who had not returned a test kit by the index date. The extra work required for the Hub and GPs to support the PEARL intervention should be evaluated and recommendations made on the feasibility of rolling out this process nationally within BCSP. The process would require refinement before being rolled out on a larger scale.


The treatment of type 2 diabetes mellitus has evolved in the present century toward safer and maybe more effective drugs, which in some cases can also reduce the risk of cardiovascular and renal outcomes. Nevertheless, we still need better strategies to reduce excess body weight in order to achieve diabetes remission, which is now a feasible target, as has been demonstrated with bariatric surgery. This review focuses on the significant advances in the management of blood glucose in type 2 diabetes mellitus, including the current understanding of the mechanisms of drug action but keeping in mind that the treatment of the disease is multifactorial. The roles of heredity and obesity were known and there were attempts to tackle the latter by changes in diet. At that time, the first-generation sulfonylureas (SUs) were developed as a successful oral replacement of insulin in most people with what we now know as type 2 diabetes mellitus (T2D). Ten years later, a study run by a university group  suggested that SUs might increase cardiovascular (CV) mortality but was harshly criticized2 and eventually disregarded with the arrival of second-generation SUs , which are still widely used. By the end of the last century, the UK Prospective Diabetes Study (UKPDS)3 demonstrated their CV safety (using chlorpropamide and glibenclamide/glyburide) but their main caveats have been the risk of hypoglycemia (particularly with glibenclamide/glyburide), increased weight, and maybe shorter duration of the effect in the long term when compared with other glucose-lowering drugs (GLDs)4. The attempt to minimize hypoglycemia by developing short-acting secretagogues such as the glinides has not been very successful.Until the middle of the last century, there was little distinction in the treatment of diabetes mellitus among people with different ages or phenotypic characteristics because only insulin was available. Although it was recognized that pancreatic failure was not the only cause, there was no other sufficient explanation5. It was also the only one which did not increase weight, and it is on the World Health Organization’s list of essential medicines. Its mechanisms of action (MOAs) are still being discussed, but it has been considered essentially an insulin sensitizer (at least in the liver).By the same time that SUs started to be used, the first biguanide (phenformin) was also launched but was withdrawn in most markets in the late ‘70s because of fatal cases of lactic acidosis. On the other hand, metformin, which was discovered even earlier and is much safer, has been increasingly used and now most guidelines consider it the preferred first-line GLD and the best partner in combination therapy. Results from the UKPDS suggested a pleiotropic effect, since the metformin arm was the only one in which the incidences of mortality and myocardial infarction were reduced in the first 10 years when given to overweight people, even though the mean difference in HbA1c against conventional treatment was lower than in the main trial with SUs or insulin6. But they also have anti-inflammatory effects that could be anti-atherogenic. Their CV safety has been under scrutiny, and in fact rosiglitazone had to be withdrawn under the suspicion of CV harm7, which since has been refuted, especially by one randomized clinical study8. The other, pioglitazone, has shown CV benefit by reducing major adverse CV events (MACE) as a main secondary endpoint in patients with CV disease9 and by reducing stroke or myocardial infarction in patients with insulin resistance and a history of ischemic stroke or transient ischemic attacks10, but the number needed to harm  may be unacceptable. Pioglitazone is still being used in a minority of patients and its main benefit remains probably in non-alcoholic steatohepatitis11. TZDs also increase weight more than other GLDs but may change the body fat composition in favor of less visceral fat12. Dual TZDs acting on PPAR gamma and alpha receptors (glitazars) could add a beneficial effect on lipids, but the clinical results have been conflicting and only a few remain in exploratory phases13. Pan PPAR receptor agonists  have been studied considering that the increased fat oxidation promoted by PPAR delta might avoid increased adiposity and weight gain, but their safety is an issue and so far these agonists serve only as a proof of concept14.Insulin resistance gradually took over the “pancreatocentric” approach as a target for the treatment of T2D, particularly at the beginning of this century when thiazolidinediones (TZDs) were introduced as the first true insulin sensitizers. They are peroxisome proliferator-activated receptor (PPAR) gamma receptor agonists predominantly in the adipose tissue, and their main MOA is reducing lipotoxicity by “stealing” free fatty acids and increasing adiponectin15. Recent studies with a delayed-release preparation of metformin have shown that although there is considerably less absorption of the drug at the level of the ileum, the efficacy is similar to the extended-release preparation, suggesting that its main action could be intraluminal by stimulating the production of glucagon-like peptide (GLP-1) and peptide YY at the level of the ileum (where the concentration of L-cells is high)16. This preparation of metformin might prove useful in renal failure, which is now a contraindication for the use of this drug.Given the complicated story of TZDs, metformin remains the only widely used GLD to treat insulin resistance. But in fact it is an insulin sensitizer only at the level of liver-reducing endogenous glucose production in comparison with TZDs, which act at both levels (liver and periphery)17. Complete remission defined by an HbA1c of less than 6% without GLD during 6 months should now be the ultimate target of diabetes mellitus control, and besides surgery there is little progress in this field. Unfortunately, surgery is not free of adverse events, and although they can be minimized by reaching the top of the learning curve and maintaining it in each surgical group, there are still complications such as stenosis, gastric fistula, gastroesophageal reflux, ulcers, intestinal obstruction, and nutritional and vitamin deficiencies.We are still left with the issue of how to reduce insulin resistance, and the only effective strategy is to reduce sufficient weight to abolish the excess of visceral fat. In fact, bariatric surgery has served as a proof of concept, since the weight loss is considerable and leads to a remission rate of up to 70% but varies depending on the type of surgery and the center219. Weight regain after any weight-losing strategy is also an unresolved problem20. Up to one-third of bariatric surgery patients regain weight, and in one study with intensive lifestyle intervention where partial or complete remission could be observed initially in almost 12% of the subjects, there was considerable weight regain after the first year and this was probably the reason why the study failed to demonstrate reduction of CV outcomes21.There is an urgent need to develop anti-obesity drugs (or devices) that approach the results of bariatric surgery. Meanwhile, surgery, particularly when done early, is being recommended by some guidelines in patients with T2D and with a body mass index of more than 30 kg/m22. For their full effect, DPP-4 inhibitors must inhibit 70 to 90% of the enzyme and this can be sustained for 24 hours with most drugs of this class. Incretin action of the GLP-1 receptor agonists can last for up to 1 week. DPP-4 inhibitors (when combined with metformin) increase blood levels of GLP-1 by twofold or threefold, and since their efficacy is similar to SUs without causing hypoglycemia or weight gain, they are becoming the preferred oral insulin secretagogues in most guidelines23. GLP-1 receptor agonists increase GLP-1 actions up to 10-fold and, at this supraphysiological level, also reduce appetite and delay gastric emptying. Clinical trials show significant weight loss, but the main caveats are cost of injection and gastric side effects (nausea and vomiting). GLP-1 receptor agonists have been divided into short-acting (exenatide and lixisenatide) and long-acting 24. The former produce better post-prandial glucose control but less weight loss. Both short-acting (lixisenatide) and long-acting (liraglutide) GLP-1 receptor agonists are now available as “fixed-ratio” combinations with basal insulin , and the main benefits are less risk of hypoglycemia and blunting of insulin-induced weight gain25. Some of these peptides are being studied for nasal or oral administration.Although improvement of beta cell function has been elusive, enhancement of insulin production in a glucose-dependent manner is now possible with the incretin effect mediated mainly by GLP-1 . The main effect of GLP-1 is to enhance insulin secretion, but it ceases when blood glucose levels reach the normal range, thus avoiding hypoglycemia. The half-life of native circulating GLP-1 is extremely short because it is degraded by the enzyme dipeptidyl-peptidase-4 (DPP-4) but can be delayed by inhibition of the enzyme (DPP-4 inhibitors) or by developing GLP-1 receptor agonists resistant to the enzyme26 and semaglutide27—went beyond proving non-inferiority to demonstrate superiority by reducing CV events. Most of the effect can be considered pleiotropic, although there was a significant difference in HbA1c and weight between the active and the placebo group, particularly with the highest dose of semaglutide. There was also an increased incidence of retinopathy complications with the latter, and this may be due to the fast drop in blood glucose levels which may affect the retinal blood supply.Both classes of incretin-mediated GLD have been shown to be safe in long-term studies designed to meet the regulatory agencies’ requirement to prove CV safety, although these GLDs should not be used in patients with a history of chronic pancreatitis because there is still controversy on this issue. Recently, two CV safety trials—with liraglutide28. That may explain why some trials have shown benefit in patients with type 1 diabetes mellitus and glucagon is now a target for new GLDs. Drugs that block the glucagon receptor may lower blood glucose but most are only in the experimental phase29. On the other hand, hybrid peptides acting on GLP-1 and glucagon receptors such as oxyntomodulin may enhance weight loss and, if the right balance can be found, might not affect the benefit of GLP-1 on glucose control30 or even improve it by adding other peptides such as GIP31.The inhibitory effect of GLP-1 on glucagon may be almost as important as the incretin effect on insulin and has raised the importance of that underestimated hormone in the physiopathology of diabetes mellitus32. Inhibitors of SGLT2 and SGLT1 are being developed, but their balance is still an issue. There is also an increase in endogenous glucose production, probably due to glucagon stimulation which can be avoided by combining them with incretin-mediated GLDs. The SGLT2 inhibitors also decrease blood pressure, but the mechanism is not clear. It may be due to the natriuretic effect, although it is short lived, or to osmotic diuresis or other MOAs33. A recent CV safety study with empagliflozin went beyond proving non-inferiority to demonstrate superiority against placebo by reducing a composite of CV events (MACE) on top of the best current treatment in patients with CV disease. The effect can be considered pleiotropic and it started very early, suggesting a hemodynamic MOA which particularly decreased the rates of hospitalization for heart failure and mortality (secondary outcomes)34. When added to standard care, it was also associated with slower progression of kidney disease and lower rates of clinically relevant renal events than was placebo35. These findings were confirmed by the results of the CV safety trial with canagliflozin, which also showed a reduction of MACE and progression of renal outcomes36.The last class of drugs being introduced for the treatment of diabetes mellitus are the sodium-glucose co-transporter 2 (SGLT2) inhibitors, which inhibit glucose and sodium reabsorption in the proximal part of the kidney tubule. Up to 60 g/day of glucose will be excreted, leading to an insulin-independent decrease in blood glucose levels and loss of calories and thereby to weight reduction. The inhibition of SGLT2 is not complete (around 50%) and is partially compensated for by an increase in the activity of SGLT1 further down the tubules37. But ketogenesis also may be a caveat since cases of normo-glycemic ketoacidosis have been reported, particularly in insulinopenic patients under stress. SGLT2 inhibitors increase the incidence of genital mycotic infections and may not be recommended when these infections are recurrent. Their glucosuric effect will become insignificant when glomerular filtration rate is low, and they are not recommended in stages 3B, 4, and 5 of renal failure. Significantly higher rates of spontaneous fractures and amputations were found in the canagliflozin study36, and the MOA that leads to these infrequent but serious adverse events and whether it is a class effect is not yet clear.There is still no full explanation for these results, and beyond the hemodynamic effect it has been hypothesized that by shunting substantial amounts of carbohydrate into the urine, there is a progressive shift in fuel utilization toward fatty substrates. The lower insulin-to-glucagon ratio favors glucose release and ketogenesis, and apparently ketones are preferred substrates for the heart and the kidney38.In conclusion, there have been significant advances in the treatment of T2D which sometimes outpace our understanding of their MOA. On the other hand, we must keep in mind that the treatment of T2D is multifactorial, as demonstrated by the Steno 2 study, one of the best clinical trials to show a very significant reduction of mortality in T2D even beyond the initial intensive intervention


The glutamine-glutamate cycle provides neurons with astrocyte-generated glutamate/γ-aminobutyric acid (GABA) and oxidizes glutamate in astrocytes, and it returns released transmitter glutamate/GABA to neurons after astrocytic uptake. This review deals primarily with the glutamate/GABA generation/oxidation, although it also shows similarity between metabolic rates in cultured astrocytes and intact brain. A key point is identification of the enzyme(s) converting astrocytic α-ketoglutarate to glutamate and vice versa. Most experiments in cultured astrocytes, including those by one of us, suggest that glutamate formation is catalyzed by aspartate aminotransferase (AAT) and its degradation by glutamate dehydrogenase (GDH). Strongly supported by results shown in  In the adult brain neurons require metabolic collaboration with astrocytes in order to synthesize glutamate, the major excitatory transmitter, and its decarboxylation product γ-aminobutyric acid (GABA), the major inhibitory transmitter. The transport of glutamate and its precursor glutamine from blood to the brain is very slow and changes in plasma concentration of glutamine have only little influence on brain glutamate content ,2. The n2 forms oxaloacetate, another TCA cycle constituent glucose during long-term incubation of cultured astrocytes (~1 nmol/min per mg protein) by measuring increase in labeled glutamine at a time when there was no glutamate in the cultures. Earlier during the incubation, when glutamate was also present, the rate was twice as high. This might reflect direct synthesis of glutamine from glutamate, consistent with the glutamate-glutamine cycle in vivo carrying both newly-synthesized glutamate and previously released transmitter glutamate after its astrocytic accumulation. A caveat is that the cells used by these authors, like those used by Pellerin and Magistretti glutamate. A similar rate of oxidative metabolism of glutamate of (5.0 nmol/min per mg protein) was found polarographically by Hertz and Hertz [z et al. ,65,66 wend Hertz  using 10nd Hertz  found a nd Hertz  had founA previous study by McKenna et al.  comparedAs described above, Westergaard et al.  and Sonn2 formation and therefore concluded that the reaction was catalyzed by GDH, a similar conclusion to that reached by Westergaard et al. [Yu et al.  who usedd et al.  and Sonnd et al. ,68, who d et al.  that thed et al. .2 formation by AOAA. Even in the absence of AOAA the rate of CO2 formation was distinctly lower that those found at 50 µM glutamate  therebyProlonged exposure of astrocytes to extremely high glutamate concentrations would only occur in exceptional circumstances in vivo such as complete depolarization or intense firing during seizure, rendering the conclusions of the importance of GDH in many tissue culture studies mainly relevant to extreme physiological to pathological conditions. This also applies to the studies by Yu, Hertz, and Schousboe cited in + to NADH during glycolysis , is followed by malate entry into mitochondria and its conversion to oxaloacetate (OAA) when NADH is re-oxidized to NAD+; (2) that during glutamate degradation the same molecule of OAA is transaminated to aspartate when glutamate, after its entry into mitochondria in exchange with aspartate is transaminated to α-KG; (3) that the generated α-KG enters the TCA cycle, which drives the series of reactions forward, (4) whereas the aspartate molecule re-enters the cytosol, where it again supplies the aspartate needed for transamination of α-KG during the synthesis of glutamate. It is critical to note that AAT-mediated conversion of glutamate to α-KG occurs in mitochondria and that the generated aspartate travels from mitochondria to cytosol via the glutamate-aspartate exchanger/AGC1/aralar (coded by Slc25a12). In contrast, AAT-mediated glutamate synthesis occurs in the cytosol, with malate derived from aspartate via oxaloacetate travelling from cytosol to mitochondria via the malate/α-KG exchanger OGC (coded by Slc25a11). AGC1  is also required for the malate-aspartate shuttle (MAS) activity needed to carry a reducing equivalent to the mitochondria following the cytosolic oxidation of malate to pyruvate  and for the pyruvate production from glucose , but for graphical reasons this is not indicated. It is therefore important that aralar is abundantly and lastingly expressed both in astrocytes obtained directly from the mouse brain by fluorescence-activated cell sorting and in well-differentiated astrocyte cultures. At least as high mRNA expression in astrocytes as in neurons was first demonstrated in the freshly isolated cells by microarray analysis [As shown in analysis . Subsequanalysis .The high expression of aralar protein and mRNA in astrocytes freshly obtained from the brain ,102 is e4+ equivalents brought into the glia by glutamate uptake, the levels of asparate and glutamate will rise and eventually prevent the use of AAT for oxidation. The association between aspartate and glutamine synthesis is supported by the observation that exposure of cultured astrocytes to 3 mM ammonia decreases the aspartate content from 7.0 to 2.6 nmol/mg protein within 30 min [In addition to a fully functioning malate/aspartate shuttle, prevention of excessive accumulation of aspartate and glutamate requires as previously mentioned that glutamine synthesis is active in the cytosol. One amino group is introduced into aspartate during mitochondrial oxidation of glutamate and returned to glutamate during its formation in the cytosol, with the two processes coupled by the transport of aspartate. It is of critical importance that the generated glutamate is further metabolized to glutamine in order to maintain nitrogen homeostasis. Without glutamine synthesis to remove the NHn 30 min . This den 30 min ,107. It n 30 min , with a 13C labeling which could address this issue more conclusively have not been published.As reviewed above several studies, primarily of astrocyte cell cultures, have proposed an important role of GDH. However they have based their conclusions on the lack of effect of AOAA, which may be misleading in the presence of substantial amounts of extracellular glutamate or glutamine. More recently glial cell cultures with knockouts or knockdowns of GDH have been studied to more directly test the roles of GDH and AAT. Frigerio et al.  found a 13C and 15N labeling methods should make such experiments possible, including establishing the use of AAT and GDH in the glutamine-glutamate (GABA) cycle under different conditions. A number of other, related characteristics of the cycle have previously been discussed elsewhere, e.g., the mechanisms triggering the activation of this cycle and its close association with neuronal rate of glucose utilization and the similar astrocytic-neuronal and neuronal-astrocytic fluxes. Both Patel et al. [+ was found to regulate pyruvate carboxylation in cultured astrocytes [+ concentrations might activate glutamate synthesis, whereas the glutamate concentration is likely to regulate glutamate oxidation, at least within a certain range , as also concluded by Michael Robinson and coworkers [It is a paradox in brain metabolic studies that astrocytes simultaneously oxidize and produce glutamate. Several authors have previously found evidence that the dual role was accomplished by the use of two different enzyme systems, GDH for oxidation and AAT for re-synthesis, and one of us (Leif Hertz) is an author of the first study claiming GDH-mediated oxidation . Most evl et al.  and we ol et al.  have deboworkers . Couplin


Bicuspid pulmonary valve disease is rare and a small numbers of case reports describe concomitant pulmonary artery dilation similar to the dilation of the ascending aorta, which is often seen in BAVs disease. We examined three cases of bicuspid pulmonary valve disease, 10 healthy volunteers and 10 patients with BAV disease but a tricuspid pulmonary valve. All participants underwent anatomical and functional imaging of the pulmonary valve, pulmonary artery, and right ventricle as well as advanced time-resolved 3-dimensional cardiac magnetic resonance imaging (4D flow) to assess the flow pattern in the pulmonary artery. All patients with a bicuspid pulmonary valve had pulmonary artery dilation and showed distinct helical flow abnormalities with increased rotational flow and increased flow displacement compared to a mild left-handed flow pattern in the healthy volunteers. Additionally, there was marked asymmetry seen in the systolic wall shear stress (WSS) pattern, with the highest values in the anterior wall of the pulmonary artery. In comparison, patients with a BAV but a tricuspid pulmonary valve had normal flow patterns in the pulmonary artery. These haemodynamic findings are similar to recent studies in bicuspid aortic disease, and suggest the importance of flow patterns in the pathophysiology of vessel dilation in both aortic and pulmonary bicuspid valve disease.Abnormal flow patterns in the aortas of those with bicuspid aortic valves (BAVs) are increasingly recognized as important in the pathogenesis of aortic dilatation but pulmonary flow patterns in bicuspid  The acquisition slab over the area of interest includes time resolved velocity encoding images in three directions and a time resolved magnitude image per slice. These images are then corrected for Maxwell effects, aliasing and eddy currents and then reconstructed for visualization. For analysis, analysis planes are placed perpendicular to the vessel of interest and hemodynamic flow changes are quantified, such as flow angle and flow displacement, calculating how much the flow jet deviates from the midline of the vessel, rotational flow , and wall shear stress (WSS) estimations based on the interpolation of local velocity derivatives. These visualization and quantification methods have been applied to BAV disease and shown, that the majority of patients exhibit a marked right-handed helical flow pattern  and 10 age- and sex-matched patients with a BAV but normal tricuspid pulmonary valve .Twenty-three prospectively enrolled participants underwent 4D flow MRI assessment including the assessment of flow patterns in the main pulmonary artery. This included three patients with a bicuspid Each subject underwent two CMR scans—one on a 1.5 Tesla system  for anatomical imaging; the second on a 3.0 Tesla system  for 4D flow assessment, both using a 32-channel cardiac coil. All images were electrocardiogram (ECG)-gated. Steady-state free-precession (SSFP) cine sequences acquired during a single breath-hold were used for pulmonary artery dimension measurements, right ventricular volume assessment and pulmonary valve morphology. The velocity across the pulmonary valve was measured using through-plane phase contrast velocity mapping in an image slice placed perpendicular to the pulmonary artery, just above the valve tips. CMR42  was used for analysis of standard anatomical and velocity parameters.3, temporal resolution 40 ms. The velocity encoding range was determined using the lowest non-aliasing velocity on scout measurements. Data acquisition lasted 10–15 min and the data was therefore collected from and averaged over hundreds of cardiac cycles. Dataset processing and WSS calculation were conducted with customized Matlab software Version R2010a  and EnSight Version 9.1.2 , as described previously  was one of the first “blue” babies that underwent catheter balloon valvotomy of his pulmonary valve in London in 1949. He has not needed any intervention since. He was a non-smoker, overweight (BMI 35) and on no cardiovascular medications but hypertensive during the study visit (169/69 mmHg). He also suffered from gout.Of the three patients with bicuspid pulmonary valve during a recent cardiovascular magnetic resonance exam. He was an ex-smoker, normotensive and on no cardiovascular medications.Patient 2 (73 years) was diagnosed incidentally with a bicuspid Patient 3 (47 years) had more complex heart disease with a double outlet right ventricle and marked pulmonary stenosis. He was an ex-smoker, normotensive, and on no cardiovascular medications. He also suffered from gout.BAV disease patients had isolated valve disease with no other cardiovascular problems such as coronary artery disease or coarctation of the aorta. The age range was 47–72 years. The patients were normotensive, 4 patients were on blood pressure lowering agents, 1 participants still smoked, and 4 participants were ex-smokers. All participants had normal left ventricular function (ejection fraction 57–70%). Two patients suffered from gastroesophageal reflux.All healthy volunteers were free from cardiovascular disease or any other non-cardiac disease. The age range was 47–75 years. They were normotensive and not actively smoking.pulmonary valve  and he had normal right ventricular (RV) function . Patient 2 also had a well-functioning bicuspid pulmonary valve  with normal right ventricular function . Patient 3 had more complex heart disease with a double outlet right ventricle and marked pulmonary stenosis . All three patients were male and had a normal aortic valve with normal ascending aortic measurements. However, they all had a dilated pulmonary artery  compared with 2.6 ± 0.3 cm in the healthy volunteers.Patient 1 had a well-functioning bicuspid 2; Figure 2. The 3rd patient had a marked left-handed helical flow  than the healthy volunteers (0.88 ± 0.25 N/m2) and only elevated in patient 3 with a stenotic (narrowed) pulmonary valve (1.39 N/m2). However, there was a marked asymmetry seen in all three patients with highest WSS values in the anterior section with a marked anterior-posterior asymmetry  compared to healthy volunteers with negligible asymmetry , rotational flow , flow angle , displacement  and mean systolic WSS  is the underlying cause for the observed abnormalities alone.The morphology of the valve appears to be central to the development of abnormal flow patterns. In patients with a BAV but a tricuspid pulmonary valve may be clinically rare, these novel findings further underline the importance of haemodynamic flow disturbances in the pathophysiology of vessel dilation in bicuspid valve disease. To assess the prognostic value of 4D flow MRI and the clinical implication of our findings, longitudinal cohort studies of both bicuspid aortic and pulmonary valve disease are necessary to understand the degree of involvement haemodynamic changes have in vessel dilation and whether 4D flow MRI is a clinically useful imaging biomarker for predicting aortic growth rate.Blood into the pulmonary artery is ejected at much lower pressures than into the aorta which may explain the only mild effects of the helical flow pattern on WSS and wall dilation. While a bicuspid pulmonary valve disease is rare, patient numbers were small. 4D flow MRI is a new imaging technique but measures such as WSS have been validated, even though the true WSS is likely to be higher than the measured value, due to limited spatial resolution and partial volume effects, as discussed previously (Markl et al., As bicuspid MB designed the work, completed the acquisition, analysis and interpretation of the work, drafted the work, approved the final version and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. ML contributed to the design and acquisition of the work, revisited the work critically for important intellectual content, approved the final version and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SN and SM supervised the design of the study and interpretation of the work, revisited the work critically for important intellectual content, approved the final version and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.