Magic roundabout (ROBO4) is an unusual endothelial-specific paralog of the family of neuronally-expressed axon guidance receptors called roundabouts. Endothelial cells (ECs), whose uninterrupted sheet delimits the lumen of all vertebrate blood vessels and which are absent from invertebrate species, are a vertebrate-specific evolutionary novelty.Herein, the evolutionary mechanism of the duplication, retention and divergence of ROBO4 was investigated for the first time. Phylogenetic analyses carried out suggested that ROBO4 is a fast-evolving paralog of ROBO1 formed at the base of vertebrates. The ancestral expression pattern was neuronal. ROBO4 dramatically shifted its expression and became exceptionally specific to ECs. The data-mining of FANTOM5 and ENCODE reveals that ROBO4’s endothelial expression arises from a single transcription start site (TSS), conserved in mouse, controlled by a proximal promoter with a complex architecture suggestive of regulatory neo-functionalization. . Further evidence for the neo-functionalization of ROBO4 comes from the analysis of its protein interactions, the rates of protein evolution, and of positively selected sites.The neo-functionalization model explains why ROBO4 protein acquired new context-specific biological functions in the control of angiogenesis. This endothelial-specific roundabout receptor is an illustrative example of the emergence of an essential vertebrate molecular novelty and an endothelial-specific signaling sub-network through 2R-WGD. The emergence of novel cell types, such as ECs, might be a neglected evolutionary force contributing to the high rate of retention of duplicates post-2R-WGD. Crucially, expression neo-functionalization to evolutionarily novel sites of expression conceptually extends the classical model of neo-functionalization. An endothelial-specific paralog of an axon guidance receptor ROBO1 was cloned and called magic roundabout, or ROBO4  sequences of ROBO3. The authors proposed that the function of ROBO3 shifted from a receptor for SLIT repulsion into a receptor that silences SLIT repulsion and enhances attraction to netrin. The mammalian-specific substitutions were implicated in ROBO3’s lost the ability to bind SLIT. We note that the observation of positively selected sites in ROBO3 is in agreement with the increased length of its branch in A model similar to  authors . Zelina et al. to ameliorate LBA in some cases.The early diverging topology of the ROBO4 branch in An alternative model in For the discussion of an alternative model see section entitled In the following sections, various aspects of the divergence of roundabout ohnologs are investigated. We start with protein interactions for which the role of gene duplications, especially the resolution of the network after WGDs, are still incompletely understood. To investigate ROBO4 from a network perspective, the STRING database of functional protein interactions was queried . The datInterestingly, the interaction network suggests that ROBO4 is a network hub (with 21 edges) and a bridge  integratD. melanogaster. However, ROBO1 and ROBO4 share some interaction partners  which is common for paralogs. Nevertheless, the majority, 12, of ROBO4’s interaction partners are not shared with ROBO1.Unlike ROBO4, ROBO1 is not a network bridge. Instead, it is embedded entirely within the axon guidance sub-network with most of interactors corresponding to the ancestral network of robo1 in the D. melanogaster. Ten interacting proteins were found: slit (sli), Abl tyrosine kinase (Abl), leak (lea), enabled (ena), netrin A and B (NetA and NetB), dally-like (dlp), robo3, commissureless (comm), and Eph receptor tyrosine kinase (Eph). Note that robo3 is a fly-specific paralog of robo, it is not a 1-to-1 ortholog of vertebrate ROBO3. Because of the fly-specific paralogs, the estimate of the ancestral network is not optimal but it is the best possible with the data available today. An alternative would be to use the network of C. elegans, where there is only one roundabout ortholog: sax3, but because of the worm’s unique development it’s signaling pathways are typically rather diverged.The key test of the neo-functionalization model for network interactions is the comparison of the networks of paralogs with the ancestral network. To estimate the ancestral pre-2R-WGD interaction network, we searched the STRING database for the interactions of robo1 in The majority ROBO4’s interactions do not correspond to the ancestral network of robo1, suggesting they were acquired after 2R-WGD, during ROBO4’s asymmetric divergence. Similar conclusions are reached if experimental interactions of ROBO4  are examF5 is not only more comprehensive than functional genomics resources available before, but it also allows the discovery of alternative transcription start sites (TSSes) at single-base resolution which has never been performed for roundabouts. Comprehensiveness is crucial when investigating tissue-specificity; if only a few cell- or tissue-types are examined, alternative sites of expression may be missed leading to false presumption of tissue-specific expression.th most enriched transcript in the group of F5 endothelial libraries . However, the ROBO1-TSS2-linked transcript was still fairly abundant in the endothelial libraries (the average of 93 TPM). The transcript of ROBO1-TSS1 had the average endothelial expression of only 10 TPM. The other roundabout transcripts were absent from ECs . We note that the endothelial expression of ROBO1 could also be interpreted as partial neo-functionalization, although this ohnolog conserved the ancestral neuronal expression site. A general increase in the rate of expression divergence of orthologs in the presence of paralogs was reported previously .P-value is calculated using a randomization test appropriate for estimating the genomic background / random expectation for a co-expression analysis , that is the highest expression level attained in any tissue- or cell-type. ROBO4-TSS1 has a very potent ME of 979 TPM . The valAs ROBO4 neo-functionalized to endothelial-specific expression, we compared the architecture of its proximal promoter with those of other roundabouts in search for clues of how this expression shift was effected. Using ENCODE data for human umbilical vein endothelial cells—HUVECs , we founNone of the other roundabout promoter had either a similar set of TFBSes or the similar structural arrangement into proximal and distal clusters as ROBO4-TSS1 promoter did. In the kA) and synonymous (kS) substitutions were calculated on different branches the roundabout tree and their ratio was used to estimate the average evolutionary rate for the entire sequence length. However, the rate of evolution over the entire length of the coding sequence is very rarely higher than one, even if the protein is positively selected. Therefore, individual sites of positive selection among the codons of ROBO4 were searched for using variable ratio [ROBO4 was included among genes under positive selection in the dolphin genome . This oble ratio  and branle ratio , 57. Theneuronal. ROBO4, in contrast, is powerfully and specifically expressed in ECs from a single TSS (ROBO4-TSS1) identified here using F5. Drosophila’s robo, the single invertebrate roundabout ortholog [The analyses performed herein prove that ROBO4 neo-functionalized on the level of gene expression. Considered as a group, vertebrate ROBO1/2 and 3 should be labeled ortholog , represeortholog , 59, ROBortholog . InsteadWhat is exceptionally interesting about ROBO4 is that its emergence was entangled with the emergence of the novel cell type: the endothelial cell. As there is no true endothelium in invertebrates , this ceet al. proved the importance of the three kilobase (upstream of the TSS) proximal promoter of ROBO4 for controlling its endothelial-specific expression [et al. used deletions, electrophoretic mobility shift assays and chromatin immunoprecipitation assays to analyze the proximal promoter of ROBO4 detecting several candidate TFBSes, including SP1 and a GA-binding protein (GABP)-binding motifs. In transgenic mice, the GABP site was shown to contribute to ROBO4’s EC expression [If ROBO4’s expression pattern is a neo-functionalization, how is it regulated? Okada pression . Okada epression .et al. focused (2007). There was a weak SP1 site (QS = 128) but it came from a carcinoma cell-line not ECs. There was no site for GABP. This discrepancy is likely due to the fact that ENCODE did not include GABP or SP1 ChIP-seq on ECs [et al.Can Okada’s TFBSes be found in ENCODE? This question is answered in q on ECs  examinedHowever, ENCODE data suggested a new definition of the architecture of the proximal promoter of ROBO4. The data also facilitated its comparison between ECs and a non-EC cell types . There wFinally, we note that DNase I signal suggested that the promoter was accessible in 66–39% of 125 ENCODE cell lines. Such open chromatin has to attract TFs and indeed many weak non-EC TFBSes overlapped endothelial ones. Such overlapping clustering of TFBSes suggests they form complexes and cooperate. It is tempting to speculate that the weak non-EC TFBSes contribute to endothelial-specificity of ROBO4’s as inhibitors of transcription which can bind the regions of open chromatin when they are not bound by endothelial-specific TFs. However, an extensive analysis of genome-wide trends would be necessary to test this hypothesis and this was not in the scope of the current manuscript.The architecture of the promoter of ROBO4 was compared against others in the genome including those of roundabout paralogs. It was found to be unique in the genome, highly unlikely to have been assembled by chance, and dissimilar to those of the architectures of other roundabout promoters B. Moreovde novo, but no further clues about its origins can be offered here. In any case, ROBO4-TSS1 almost certainly is not homologous to any of the other TSSes of roundabouts.Could ROBO4 have acquired an entirely new promoter after duplication, down-stream of the original TSS? This is strongly suggested by the fact that the ROBO4 protein is N-terminally truncated in relation to the alignment of other roundabouts . The expression of ROBO4 in endothelial cells is a clear neo-functionalization on the level of gene expression. We can speculate that ROBO4 neo-functionalized to its endothelial-specific expression because of the powerful adaptive advantage conferred by the pressurized circulatory system which ROBO4 helped to shape and which emerged through 2R-WGD. In terms of the protein interaction network, ROBO4 functions as a bridge integrating several functional sub-networks of the mammalian cell, with a change of ligand to the hUnc5b receptor which is also expressed in endothelial cells . Thus, RThe amino-acid sequences of vertebrate roundabouts were obtained from GenBank  were removed. Such duplication nodes are never genuine. The method used by TreeBeST requires the assumption of the known species tree; we used the species tree provided as the part of the TreeBeST package version 1.9.2 . This spMus musculus , Felis catus , C. lupus familiaris , Bos Taurus , Equus caballus , X. tropicalis  and Danio rerio . The lesser number of one-to-one orthologs in Danio rerio is probably a consequence of a fish-specific whole genome duplication [To estimate the probability of the conservation of a two-gene cluster analogous to ROBO3-ROBO4 we analyzed ENSEMBL orthologs. The orthologs of human genes were downloaded from the Ensembl Genes 89 BioMart  to a locaamodelpr set to mixed (where ten different amino-acid substitution models are sampled according to their posterior probabilities). The rates parameter sets the model for the variation of evolutionary rates among amino-acid sites: equal — no variation; gamma — the rate of a site is drawn from a gamma distribution; invgamma — a fraction of sites is invariable while others come from a gamma distribution. Those trees were shown in the To test the robustness of the phylogeny of roundabouts, we calculated Mr. Bayes consensus trees (50% majority rule) with varying analysis parameters. All trees were calculated for the amino-acid sequences of either: (W) whole protein, (E) extra-cellular portion, or (I) the intracellular portion of roundabouts. Trees were calculated with the F5 used CAGE technology to construct an atlas of gene expression in the human genome at a single-base resolution level . F5 humahttp://fantom.gsc.riken.jp/zenbu/). ZENBU was developed specifically to visualize F5 data in genomic coordinates [rle normalized, and filtered (such that clusters of 3 or more tags per library are shown). For expression data, either rank-sum enrichment was calculated, or the number of tags normalized per million tags in the library. Rank-sum enrichment was calculated by a Wilcoxon-mann-whitney rank-sum enrichment algorithm. The algorithm returned a z-score that was proportionate to the enrichment of expression in the sets of samples grouped in different ontologies.rdinates . All datTo calculate the relative expression of human roundabout TSSes in endothelial versus non-endothelial cells, 28 endothelial F5 libraries were identified among the total of 513 primary cell F5 libraries. The endothelial libraries included aortic, arterial, micro-vascular, thoracic, vein, umbilical vein, hepatic sinusoidal, glomerular, and lymphatic endothelial cells . In this sub-analysis, F5 tissues and cancer cell lines were not included as: (1) bulk tissues contain an infiltrating fraction of endothelial cells, (2) tissue-specificity in immortalized and transformed cancer cell lines can be altered. Preferential endothelial expression was the ratio of average expression in the endothelial over the non-endothelial subset.P-values were calculated with the core R function cor.test. The parameter method set to “pearson”. PCC was used because we previously found it works well for tissue-specific genes and in the context of the analysis of gene duplicates [rho and Kendall’s tau.To test for the co-expression of ROBO4 with other roundabouts C, Pearsoplicates . In our P-value accompanying the PCC of a pair of co-expressed genes may not be reliable as cor.test uses a t-distribution assuming that samples are normally distributed. Therefore, alternative P-values were calculated using sampled randomization. In this approach, the assignments of expression values to all RefSeq transcripts were permuted for each sample of expression. After each of the 1000 permutations, a sample of 1,000 RefSeqs was selected at random and PCCes were calculated for all possible pairs in the subset excluding self-comparisons. The distribution of the PCCes obtained through the above permutations defined an empirical cumulative distribution function. Percentiles of this distribution calculated for the PCCes from the comparisons of roundabouts defined randomization P-values displayed in However, the STRING is a bioinformatics resource where protein interactions are inferred from five lines of evidence: (1) genomic context, (2) high-throughput experiments, (3) co-expression, (4) text mining, and (5) other databases such as BioGRID . Versionhmmscan from the HMMER package v3.1b2 was used to test roundabout protein sequences against the database of 16,306 hidden Markov models of protein domains from the Pfam database release 30. E-value, that is the expected number of random hits of equal strength, was calculated by the program hmmscan from the HMMER v3.1 package [hmmscan hits . For this reason, only a single lowest scoring domain was reported for each region of the protein query in i.e. the reported domains are non-overlapping).The program  package . Only hmcan hits  with theThe ENCODE dataset included data for 161 TFs and 91 human cell types under various treatment conditions . This inThe ENCODE dataset was also downloaded for offline analysis as a BED file . The BEDImportantly, ENCODE TF binding sites (TFBSes) are assigned a quality score (QS) which can be used to divide them into strong (QS > 500) and weak sites (QS ≤ 500). Unless otherwise stated, only the strong ENCODE sites were used in the analysis.The distribution of the sizes of promoter architectures  is not normal .To estimate their divergence, promoter architectures were first encoded as vectors in 161 dimensions. The number of dimensions corresponded to the number of TFs assayed by ENCODE. Next, Manhattan distances between each pair of vectorized promoter architectures were calculated A. To putThe choice of the metrics should be defended. Manhattan rather than Euclidean distances were used as the latter have counter-intuitive properties in multidimensional spaces . AnotherSome promoter architectures may be more frequent than others simply because the probability of the occurrence of respective TFBSes in promoters is high. Other architectures may be rare; the random chance of finding given arrangement of TFBSes in the same proximal promoter may be extremely low. Rare promoter architectures may be speculatively interpreted as more informative.TFBSk,k = 1,…,161). Thus, each random variable TFBSk corresponded to counts of individual TFBSes from the merged ENCODE data. The expected probability of a given binarized promoter architecture (PE) with n-TFs was calculated assuming independence between the sites and was a product of marginal probabilities of all 161 ENCODE TFBSes. PE could be expressed in the following closed-form:k equaled one for TFs which were present in the promoter architecture (that is TFs with indices from 1 to n) and zero for TFs which were not present (that is TFs with indices from n+1 to 161).To estimate the degree of non-randomness associated with the promoter architectures of roundabouts, we calculated joint probability of each of them as an event in the multivariate probability space composed of 161 discrete random variables (O) was simply a ratio of transcript which carried such an architecture in the total population of RefSeq transcripts.The observed probability of a given promoter architecture . The ratio of PO/PE is the measure of enrichment of a given combination of TFs in real data versus random expectation. Again, to put the values obtained for roundabouts in context, we compared them against the distributions of PE, PO and PO/PE for all RefSeq promoter architectures . The following Newick format tree assuming 2R-WGD phylogeny of roundabouts was used:The was used . Codeml “ #1,  ), ,  ) );”. This tree intentionally select the branch of ROBO4 before the divergence with the murine ortholog as foreground. Any period of accelerated evolution or positive selection would be expected immediately after 2R-WGD rather than during the diversification of mammals. 2R-WGD tool place more than 450 million years ago (MYA). The evolutionary split between human and mouse occurred 65–75 MYA [5–75 MYA .d.f. equal to difference in d.f. between the two models. If the percentile was lower than the critical value of 5%, the difference was statistically significant at the P-value of 0.05.The alternative models of evolution were evaluated using the likelihood-ratio test and the chi-square test of significance following standard protocols , 100. ToBayes empirical Bayes, otherwise known as the BEB test  was usedThe conservation in mouse of each human roundabout TSS was assessed. For each 6kb promoter region in the human genome linked with a roundabout TSS, genomic nucleotide sequence was retrieved and used as a BLAT search query against the mouse assembly (mm9). The location and score of the top mouse genomic hit is given in P-value was the fraction of randomization runs where the cardinality of the intersection of the reduced promoters was equal or lesser than the observed value. The runs were repeated 10,000 times. The simulation was implemented in R.Two types of simulation models were constructed. In the first model (labeled independent-TFs), the presumed ancestral architecture was the union of the sets of TFs bound by both paralog promoters. TFBSes were treated as independent sites with the same probability of the loss. In the second model (labeled clustered-TFs), the ancestral architecture was a 6kb simulated DNA region with all the TFBSes of both paralogs . TFBS losses were simulated as cumulative cycles of random deletions of 100 basepair fragments of the DNA of the simulated ancestral promoter. Thus, TFBSes were not independent but clustered as in the original paralog promoters. In both the above simulation models, TFBS deletions in the simulated ancestral architecture were repeated until the algorithm arrived at a pair of promoter architectures of cardinalities equal or lesser than the cardinalities of the input paralog promoters. After each random run, the number of TFBSes in common between resulting paralogs was established by set intersection on their promoter architectures and compared with the observed value. The S1 FigC. savignyi or C. intestinalis. This is probably because the roundabout receptor evolved in bilaterians to sculpt their complex nervous systems necessary for movement and active search for food. As tunicates reverted to a stationary lifestyle, roundabouts became dispensable. Sea lamprey (Petromyzon marinus) has ROBO3 and ROBO4 orthologs, but they are located on short contigs which does not allow for the evaluation of synteny. Note that the tree is not to scale. The topology reflects the Ecdysozoa hypothesis [Vertebrate roundabouts are located in two tail-to-tail clusters that are conserved . In contpothesis .(PDF)Click here for additional data file.S2 Fighttp://genome.ucsc.edu/). For the TF track, shades of grey signify the QS-score of the TF binding site (the darker the hue the higher the QS). Note that there are no binding sites in the three kilobases downstream of the TSS (only polymerase type II sites—marked as Pol2). ChIP-seq peaks from HUVECs, which are the most significant for endothelial expression, are highlighted in red. There are two clear clusters of TFBSes: proximal and distal, with a CTCF site between them. Both these clusters have AP1 DNA binding motifs (highlighted as green bars) for the AP-1 hetero-dimer (consisting of JUN and FOS). There are also three CTCF-binding canonical DNA motifs and multiple CTCF ChIP-seq peaks (but only two out of four in HUVECs). Below the TFBS track, there is a wiggle-track with base-wise phyloP 100-vertebrate-genomes conservation scores [In panel (A), a window of 6 kbps around the TSS of ROBO4 was visualized online using the UCSC Genome Browser Click here for additional data file.S3 FigThis figure shows additional roundabout promoters shown in the layout analogous to the (PDF)Click here for additional data file.S4 FigThe figure shows a phylogenetic tree for a TreeFam family with accession number TF351053.(PDF)Click here for additional data file.S1 FileThis file contains a number of supplementary results: (1) on the conservation of roundabout clusters, (2) an alternative evolutionary model for the emergence of the roundabout cluster, (3) an analysis of expression patterns in genomic sub-regions neighboring the two roundabout clusters, (4) the simulation tests of the DDC model, and (5) the analysis of promoter probabilities. The file also contains two supplementary tables. The first table is devoted to the analysis of expression profiles in the genomic locus of ROBO4 (Table A). The second table is devoted to the probabilistic analysis of the promoter architectures the TSSes associated with roundabouts (Table B).(DOCX)Click here for additional data file.S2 FileThis file contains the alignment of the sequences of roundabouts.(TXT)Click here for additional data file.S3 FileThis file contains the original TreeBeST NHX-file for the phylogenetic tree of roundabouts.(NHX)Click here for additional data file.S4 FileDefault TreeBeST package v1.9.2 species tree.(TXT)Click here for additional data file.S5 FileThis file contains Mr. Bayes consensus trees with varying analysis parameters.(DOCX)Click here for additional data file.S6 Filehmmscan hits in the protein sequences of roundabouts.This file contains a list of (TXT)Click here for additional data file.


Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis . EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.Secondary cell wall (SCW) deposition in  Fast-growing angiosperm trees such as Eucalyptus are widely grown as short-rotation lignocellulosic feedstocks for pulp, paper and other renewable biomass products derived from the SCWs within wood4. The deposition of SCWs, which consist primarily of cellulose, hemicelluloses and lignin, is regulated by a complex semi-hierarchical transcriptional network composed mainly of NAC (NAM/ATAF/CUC) and MYELOBLASTOSIS (MYB) transcription factors (TFs)8. Among the known master regulators of SCW formation in the herbaceous model Arabidopsis thaliana (Arabidopsis), Secondary Wall NACs (SWNs9) appear to initiate SCW deposition through this regulatory network, occupying the upper network tier and regulating several mid- and lower-tier TFs as well as core SCW biosynthesis genes. The SWNs directly regulate, among others, the expression of several key MYB TF genes, with MYB46 and its functionally redundant homolog MYB83 also being considered master regulators positioned mid-tier, as is the CCCH-type zinc finger C3H1414.Secondary cell walls (SCWs) equip plants with pathogen resistance, mechanical support and the ability to effectively transport water from the roots to the aerial organs24. Thus, VND6 is a key regulator of the reticulated and pitted wall patterning observed in secondary xylem vessels, the deposition of which is determined by the bundled microtubule structure of the cytoskeleton26. In woody angiosperms such as Populus the expression of NST and VND clade homologs appear to overlap somewhat, with both the SND1 homolog PtrWND1B (a Populus trichocarpa wood-associated NAC domain protein) and the VND7 homolog PtrWND6A being expressed in xylem and phloem fibres, while vessel-specific differentiation appears to be regulated by the exclusive expression of PtrWND6A in vessels28. High-resolution spatial transcript profiling in aspen from phloem through the cambium to the lignified xylem zone revealed biphasic expression peaks for SND1 homologs in phloem and early xylem SCW deposition, while VND6 homolog transcripts peaked either during xylem SCW deposition or its cessation29, suggesting a specialization of SND1 homologs in phloem and xylem fibre formation and VND6 homologs in vessel differentiation. However, dominant repression in Populus of either the SND1 homolog PtrWND2B or the VND7 homolog PtrWND6B resulted in significantly reduced xylem SCW deposition in both fibres and vessels30. Although this suggests less distinct roles for SWN-mediated regulation of fibre and vessel SCW formation in woody angiosperms, the question of partially overlapping versus distinct roles in secondary xylem development remains poorly resolved. For example, homologs of SND1 and VND6/VND7 in monocots (a lineage lacking secondary xylem derived from a vascular cambium) appear to be expressed indistinguishably in sclerenchyma fibres and vessels32, while in Norway Spruce (a woody gymnosperm lacking fibres and vessels) sufficiently distinct roles could be inferred for VND and NST homologs during xylogenesis33.Arabidopsis SWNs in the NST clade, among them NST1 (NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1), NST2 and SND1 (SECONDARY WALL-ASSOCIATED NAC DOMAIN THICKENING FACTOR 1), regulate SCW formation in fibres, anther endothecia and silique valves to a large degree of redundancy, while VND (VASCULAR-RELATED NAC DOMAIN) clade TFs encompassing VND1 through VND7 are vessel-specific, with VND6 specifically regulating metaxylem SCW deposition and VND7 regulating both meta- and protoxylem vessel formation35. Congruent with their proposed functions in xylem vessel development, VND6 and VND7 strongly activate PCD genes in Arabidopsis36. PCD and autolysis, which are distinct biological processes, are initiated by Ca2+ influx signals resulting from extracellular proteolysis by serine proteases38. Proteins currently known to be involved in vessel autolysis include XYLEM CYSTEINE PEPTIDASE 1 (XCP1), XCP2 and METACASPASE 9 (MC9) that together cause autolysis of the cytoplasm, the endonuclease ZEN1, XYLEM SERINE PEPTIDASE 1 (XSP1), and possibly ARABIDOPSIS THALIANA SUBTILASE 1.1 (SBT1.1)44. The later onset of PCD in fibres may be attributed to the weaker activation of PCD genes by SND1 and slower accumulation of the necessary enzymes to prompt cellular degradation46. Compared to fibres, metaxylem vessels exhibit extensive, pronounced pits in their SCWs that allow for the lateral movement of water to living fibres and xylem parenchyma48. Pits are established by the recruitment of microtubule disassembly proteins MIDD1 and Kinesin 13 A by pit localization of GTP-bound ROP11, a Rho GTPase that is localized to oval planar domains at the plasma membrane by IQD13, activated by ROPGEF4 and deactivated by ROPGAP351.Xylem fibre and vessel differentiation is distinguished by differences in SCW thickness and patterning as well as the timing and rate of programmed cell death (PCD) and autolysis, which in the case of water-conducting vessel elements yields hollow lumens shortly after SCW deposition52, the prevalence and significance of lineage-specific adaptations to the SCW transcriptional network architecture among angiosperms are poorly understood. In Populus, for example, numerous co-orthologs of SND1 exist, of which alternative splicing of PtrWND1B in particular produces full-length and intron-retaining, truncated isoforms of the protein that can form non-functional heterodimers55. Since Arabidopsis does not harbour known SND1 splice variants, this shows that SND1-mediated SCW regulation may differ between plant lineages. In the Eucalyptus woody model, a small number of studies have aided in our understanding of SCW regulation61. To date, our knowledge of the functions of SWN master regulators in Eucalyptus is limited to a few gene targets of EgWND1, an E. gunnii homolog of SND130, with some evidence that an intron 2-retaining splice variant may also exist in E. grandis27. In previous work62, we identified putative E. grandis orthologs of SND1  and VND6 (EgrNAC26) which we hypothesised regulate fibre and vessel SCW development, respectively.Despite what is believed to be substantial evolutionary conservation in SCW transcriptional regulation63. The development of high-precision multipartite synthetic gene constructs, however, assumes individual parts possess modularity  and orthogonality , both of which are poorly demonstrated for SCW TFs. For instance, what gene targets might we expect to be activated when SCW TF homologs from diverged lineages are heterologously and ectopically induced, and how is this influenced when their endogenous promoters are exchanged with synthetic promoters? In this study, we functionally characterized E. grandis SCW master regulators through observation of their SCW network targets over time in the context of Arabidopsis mesophyll protoplasts, and their promoter activities in woody and non-woody models. Our results show that EgrNAC61 is a more potent activator of SCW genes while the VND6 homolog EgrNAC26 mainly regulates PCD and autolysis in vessels.While functionally characterized SCW TFs and their promoters are potential tools for the re-engineering of woody biomass, their crude manipulation generally results in undesirable phenotypes and hence refined approaches adopting synthetic and systems biology are favourableEgrNAC26 and EgrNAC61 coding sequences from E. grandis developing secondary xylem cDNA for cloning. We found no evidence of alternative splice variants among the amplicons  libraries, were within the top percentile of the detected transcript levels and expressed at similar levels across time points and replicates  and late (likely indirect) gene targets of EgrNAC26 and EgrNAC61 in a heterologous background, we transiently overexpressed these genes in linkage with a dual GFP reporter cassette in Arabidopsis leaf mesophyll protoplasts. Total RNA was isolated 7 and 14 hours post-transfection (hpt) from three independent protoplast isolations per time point. Transfection efficiencies were approximately 30–50% Fig. . Followites Fig. . Principtes Fig. , suggestEgrNAC26 (EgrNAC26-OX) and EgrNAC61 (EgrNAC61-OX) followed by FPKM and fold-change filtering  identified 276 , 1,022 , 685  and 1,771  differentially expressed genes (DEGs), of which the vast majority were upregulated , only one transcript was alternately up- or down-regulated across time points, and DEGs common to EgrNAC26-OX and EgrNAC61-OX datasets were all up- or down-regulated concordantly, demonstrating robust and consistent activation of a subset of shared genes. Overrepresented biological processes were identified for EgrNAC26-OX and EgrNAC61-OX DEGs through Gene Ontology (GO) enrichment analysis. Significantly overrepresented terms among up-regulated DEGs in the EgrNAC26-OX and EgrNAC61-OX datasets were largely associated with SCW biosynthesis, particularly at 14 hpt. Common terms included secondary cell wall biogenesis, cellulose biosynthesis and (glucurono)xylan biosynthesis  insight into the possible roles of EgrNAC26 and EgrNAC61 SWNs in fibre and vessel SCW regulation in eucalypts, (2) evidence of considerable conservation in SWN gene targets between Eucalyptus as a representative of the basal eudicot lineage the Myrtales which divergence from the Brassicales over 100 million years ago69, and (3) a demonstration of the regulatory dynamics and specific targets of orthologous master regulators associated with fibre and vessel differentiation in a heterologous context typical of a synthetic biology strategy.In this study, we explored the temporal transactivation dynamics of Arabidopsis genes  by homologs of SCW master regulators of fibre and metaxylem vessel formation from 70. These previous investigations revealed preferential activation of PCD-associated genes such as XCP1 and XCP2 during vessel differentiation by VND6 and VND7. In woody angiosperms, only one study has reported the genomic targets of a fibre-associated SCW master regulator, in this case a time-course experiment of poplar stem differentiating xylem protoplasts transfected with Ptr-SND1-B1, a homolog of SND1 from P. trichocarpa71. Ours is the first study to probe the global transcriptional targets of EgrNAC26 and EgrNAC61, albeit in a heterologous system (Arabidopsis). One advantage of our approach is that the transfections for each construct were performed in parallel from a single protoplast preparation, allowing for a direct comparison of the gene targets and temporal activation dynamics of each SWN candidate. Two limitations should be noted: first, mesophyll protoplasts may not reflect the accessible chromatin landscape or contain the same co-factors as xylem mother cells. However, SWNs and their homologs are known to be particularly powerful SCW master regulators, sufficient for ectopic tracheary element transdifferentiation in many cell types74. The inherent transcriptional transactivation properties of EgrNAC26 and especially EgrNAC61 in yeast  consists of lignified and suberized cells that undergo PCD and have cell walls in which a guaiacyl-rich lignin backbone predominates76. Phloroglucinol staining revealed small amounts of lignin in the phellem of the poplar transgenics , VND2 and TRACHEARY ELEMENT DIFFERENTION-RELATED 7 (TED7). LBD30/ASL19 and LBD18/ASL20 are targets of VND6 that participate in a positive feedback loop during vessel differentiation77, and VND2 is a vessel-specific SCW master regulator that interacts with VND774. TED7 is a vessel-specific protein with an unknown role in tracheary element SCW formation, acting with TED6 and interacting with the SCW-related CesA complex78. While TED6 was significantly upregulated by both EgrNAC26 and EgrNAC61 at 14 hpt  EgrNAC26 activated proteins associated with SCW pit formation in vessels, uniquely among them RHO GUANYL-NUCLEOTIDE EXCHANGE FACTOR 4 (ROPGEF4) and MICROTUBULE-ASSOCIATED PROTEIN 70-5 (MAP70-5)  EgrNAC26 transfection induced stronger activation of the chief genes associated with autolysis of tracheary elements during PCD, namely XCP1, XCP2 and MC946, as well as BIFUNCTIONAL NUCLEASE 1 (BFN1), the closest homolog of the Zinnia endonuclease ZEN1 known to degrade nuclei in tracheary elements41  EgrNAC26 transfection uniquely induced vessel-specific regulators of SCW deposition, among them LBD30/ASL9, VND2 aLBD30/ASL9, VND2 aLBD30/ASL9, VND2 aC26 Fig.  strengthLBD30/ASL9, VND2 aEgrNAC26 in protoplasts was substantially lower than that of EgrNAC61, despite the use of the same constitutive promoter and the fact that the transfection efficiency for EgrNAC26 was somewhat higher , PtrMYB020 (MYB83), PtrMYB128 (MYB103), PtrNAC156 (SND2), PtrKNAT7 (KNAT7) and PtrMYB28 (MYB58/MYB63) more strongly than VND clade SWNs55, a trend that also emerged in our time-course experiment where SCW structural genes were preferentially activated by EgrNAC61 much earlier and to a stronger level than for EgrNAC26 , 3,660 bp  and 1,115 bp  directly upstream of the translational start site were PCR-amplified from genomic DNA (Table ® (EgrNAC61) or pENTR/D-TOPO® (EgrNAC26 and VND6) entry vectors  and transferred to pBGWFS785 using the Gateway LR ClonaseII Enzyme Mix (Invitrogen). For Populus GUS constructs, the promoters were transferred to pMDC16286. Developing secondary xylem RT-PCR products of EgrNAC26 and EgrNAC61 coding regions were cloned into pCR8/GW/TOPO® and transferred to the pUC-35S-Rfa-35S-GFP protoplast expression vector71 and pDEST-GBKT7 yeast expression vector87 similarly using LR recombination. Empty vector controls of the expression vectors were produced through LR recombination with a self-ligated pCR8/GW/TOPO® vector. Large-scale plasmid isolations for protoplast transfections were performed using the PureLink HiPure Plasmid FP Maxiprep Kit (Invitrogen), or caesium chloride purification88. The pHBT::sGFP(S65T)-NOS vector89 was used as a fluorescent marker to assess the effect of different plasmid purification methods on protoplast transfection efficiency.Promoter fragments of 2,009 bp  were transformed into Arabidopsis thaliana using the Agrobacterium-mediated floral dip method90 and the transformed lines were selected on half-strength MS agar medium using Glufosinate-ammonium (20 mg/l). Transgenic Arabidopsis seeds were surface-sterilized, plated on nonselective nitrate-enriched media and allowed to germinate for 2–3 weeks. For poplar transformation, the EgrNAC61pro::GUS construct in the pMDC162 backbone was introduced into poplar hybrid Populus alba × P. tremula 717-1B4 by co-cultivation of Agrobacterium solution with leaf discs followed by shoot regeneration on selective media containing 30 mg/l Hygromycin. Transgenic poplar plants were propagated using greenwood stem cuttings and young developing stems (internodes 1 and 5) were harvested. GUS staining was performed as described by Spokevicius et al.91, excluding acetone treatment, but adding 0.5 mM potassium ferrocyanide and 0.5 mM potassium ferricyanide. After incubation at 37 °C overnight, seedlings were destained in 70% ethanol and visualized with differential interference contrast and brightfield microscopy.The pBGWFS7 constructs  through lithium acetate/polyethylene glycol-mediated transformation as previously described92. Transformed colonies were screened on media lacking tryptophan (Trp-), media lacking tryptophan and histidine (Trp- His-), as well as Trp- His- media supplemented with 1 mM or 15 mM 3-amino-1,2,4-triazole . Trp- His- media was prepared using -Trp-Leu-His drop-out supplement (Clontech™) and adding the appropriate amount of leucine supplement (Sigma-Aldrich®). Colonies were incubated for 3 days at 30 °C.The pDEST-GBKT7 constructs containing A. thaliana ecotype Columbia (Col-0) seedlings were grown for four weeks at 22 °C under a 10 h photoperiod prior to mesophyll protoplast isolation from leaves as previously described93. Multiple plants were pooled for protoplast isolations and de-bulked to form three independent biological replicates. Large-scale transfections with pUC-35S-EgrNAC26-35S-GFP, pUC-35S-EgrNAC61-35S-GFP, and pUC-35S-NOS-35S-GFP (empty vector) constructs were conducted using 4 × 105 protoplasts for each of three technical replicates per biological replicate (n = 3) to account for transfection variation and incubated for 7 h and 14 h at room temperature in the dark. Technical replicates were bulked and harvested through centrifugation at 500 × g. Of the harvested cells, 10 μl was used to determine transfection efficiency (at 14 hpt only) and the remaining cells were flash-frozen and stored at −80 °C.94. The RNA-seq reads were formatted with FastQGroomer95 prior to mapping to the A. thaliana genome (TAIR10) using TopHat 296. The mapped RNA-seq reads were assigned to gene models and the fragments per kilobase of transcript per million reads mapped (FPKM) values for each mapped transcript was calculated in Cufflinks97 using the A. thaliana TAIR10 annotation. The list of genes generated by Cufflinks was compiled into a single list with all nine libraries and Principle Component Analysis (PCA) was applied in R98 using the prcomp function99. Differentially expressed genes (DEG) were identified using the mapped RNA-seq reads in Cuffdiff97, with FPKM ≥1, false discovery rate (FDR) >0.05 and fold change ≥2 or ≤0.5.Total RNA was isolated using the Qiagen RNeasy Mini Kit  and RNA quality was determined using an Experion instrument (Bio-Rad). Stranded 100 nt paired-end (PE100) mRNA sequencing was performed at Beijing Genomics Institute  on the Illumina HiSeq4000 platform using fragment sizes of up to 200 bp. Raw RNA-seq reads passing initial quality filtering by BGI were further assessed for quality with FastQC101 was used to determine overrepresented Gene Ontology (GO) terms in the up- and down-regulated EgrNAC26 and EgrNAC61 DEG datasets. A hypergeometric test with Benjamini and Hochberg FDR correction with a P-value cut-off of 0.05 was applied with the whole A. thaliana annotation as a reference set.The BiNGO plugin in Cytoscapehttps://www.ncbi.nlm.nih.gov/sra), accession SRP117192.The RNA-seq data reported in this study is freely available at the NCBI Sequence Read Archive (Supplementary informationDataset 1


Bombyx mori, a typical representative of metamorphic insects, is of great agricultural and economic importance. The steroid hormone ecdysone  is the central regulator of insect developmental transitions, and its nuclear receptors are crucial for numerous biological processes, including reproduction, metabolism, and immunity. However, genome-wide DNA regulatory elements and the ecdysone receptor (EcR) that control these programs of gene expression are not well defined.The silkworm, In this study, we investigated the alterations in three types of histone modification in silkworm embryonic cells treated with 20E by chromatin immunoprecipitation sequencing (ChIP-seq). We identified enhancers using histone modifications and derived genome-wide ecdysone-dependent enhancer activity maps in the silkworm. We found enhancers enriched for monomethylation of histone H3 Lys4 (H3K4me1) that showed dynamic changes in acetylation of histone H3 Lys27 (H3K27ac) after 20E treatment and functioned to regulate the transcription of specific genes. EcR regulated transcription by binding not only to proximal promoters but also to the distal enhancers of target genes. Moreover, only 52.65% EcR peaks contained ecdysone response element (EcRE) motif, suggesting that EcR regulates the expression of target genes not only by binding directly to EcRE, but also by binding with other transcription factor.Our findings provide novel insights into the complex regulatory landscape of hormone-responsive cell activity and a basis for understanding the complex transcriptional regulatory processes of ecdysone.The online version of this article (10.1186/s13072-018-0216-y) contains supplementary material, which is available to authorized users. Spatial–temporal specificity in gene expression is achieved according to information in cis-acting DNA regulatory elements including insulators, promoters, and enhancers , 2. EnhaPost-translational modifications of histones, such as acetylation and methylation of lysine residues of histone H3, play important roles in regulating gene expression by altering the chromatin structure . Genome-Drosophila [Although enhancer elements are known to be associated with certain histone modifications, the relationship of these modifications with gene expression has not been clearly defined –19. H3K4osophila , 22. TheBombyx mori, a typical representative of metamorphic insects, is of great agricultural and economic importance [Hsp27, Eip28/29, Fbp1, and Sgs-4 are induced directly by ecdysone and contain an EcRE in their basal promoter region [The silkworm, portance . The steportance –28. The portance –33. The portance , 34. Theportance . Howeverr region –38. The r region . While tr region , the mecIn this study, we carried out chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies for EcR and three different histone marks to identify ecdysone-responsive regulatory elements, using silkworm embryonic (BmE) cells treated with 20E. We then correlated the histone profiles with genome-wide gene expression levels obtained by RNA sequencing (RNA-seq), to infer functional states of histone modifications and to link the regulatory elements to their target genes. We expected this integrative analysis to provide novel insights into the complex regulatory landscape of hormone-responsive cell activity.To investigate genome-wide alterations in histone modifications and to identify ecdysone-responsive enhancers in silkworm BmE cells, we performed ChIP-Seq using antibodies against H3K4me1, H3K4me3, and H3K27ac before and after 20E treatment. Previous studies have shown that H3K4me3 is an active mark that is often found in the proximal promoter region, while H3K4me1 is generally associated with enhancers located in the distal promoter region , 19. H3KWe determined the distribution of each histone modification at the genome level Fig. a. Of theComposite profiles of each histone modification around the transcription start site (TSS) were generated according to the expression level of the corresponding gene Fig. b. H3K27aHR3 locus . These results suggested that H3K27ac changed more dynamically after 20E treatment, and the activity-dependent increase in H3K27ac at the HR3 locus might be required for HR3 transcription.To examine whether histone modification statuses were altered throughout the genome by treatment with 20E, we firstly report the overlap of called peaks between 20E-treated BmE cells (“Treated”) and DMSO-treated BmE cells (“Control”). The results showed that about 60% of H3K4me1 sites overlapped before and after 20E treatment, and corresponding to 95.4% of H3K4me3 sites and 84% of H3K27ac sites. It’s worth noting that the number of altered H3K4me1 sites was similar to altered H3K27ac sites. Besides, histone modification signals were searched between 20E-treated and DMSO-treated BmE cells, and the differences in signals based on direct overlap of called peak were evaluated  is an early ecdysone-responsive gene encoding a family of zinc-finger transcription factors, which play an important role in the metamorphosis of insects  analysis  were located in a region that contained at least one EcRE motif  supplemented with 10% fetal bovine serum at 27 °C. Unless stated otherwise, we used 20E  at a concentration of 41 mM and an incubation time of 24 h.6 BmE cells were treated with DMSO or 20E for 24 h in triplicate. Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol and was treated with DNase I. Purified RNA was prepared for sequencing with the Illumina mRNA-seq Sample Prep Kit. Samples were sequenced on an Illumina HiSeq 2500 (150-bp paired-end reads). For each sample, more than 25 million clean reads were obtained.Approximately 10http://sgp.dna.affrc.go.jp/index.html) [The quality of the raw and processed reads was evaluated using FastQC (version 0.11.6). Raw data were processed using Trimmomatic (version 0.36) to remove reads containing adapter sequences and low-quality reads . We usedex.html) . We retaex.html) . We inteex.html) . Differe8 BmE cells were treated with DMSO or 20E for 24 h. ChIP-seq was performed as previously described with minor modifications [Approximately 10ications . Brieflyhttp://meme-suite.org/) and RSAT peak-motifs with default parameters [http://jaspar.genereg.net/).ChIP-seq data were pre-processed similar to RNA-seq data. We used bowtie2 (version 2.2.9) to map the paired-end clean reads to the silkworm reference genome downloaded from KAIKObase . Only unrameters . CompareThe regions with altered signals of histone modifications induced by 20E were identified by comparing the ChIP-seq tag counts between the 20E-treated and DMSO-treated BmE cells. The differences in signals based on direct overlap of called peak were evaluated by DEseq2.To determine the locations of H3K4me1, H3K4me3, H3K27ac, and EcR peaks, we firstly divided the whole genome into five unique regions according to gene set annotations , TTS and intergenic region). Then, we assigned each peak to these regions. For some peaks that overlapped with several annotation regions, only peak summit sites were considered.We used H3K4me1 sites to define enhancers according to a previous study . ExcludiBglII and HindIII with the core hsp70 promoter. Enhancers varied in size, and we used only approximately 1-kb regions around the center of enhancers as candidate enhancers. Candidate enhancers were PCR-amplified from silkworm genomic DNA, cloned into the reporter vector, and verified by Sanger sequencing. The primers used to amplify candidate enhancers are listed in addition files  were transfected with 110 ng of DNA, including 10 ng of Hrp3 vector as a transfection control, using X-tremeGENE HP DNA Transfection Reagent (Roche). After transfection for 24 h, the BmE cells were treated with 20E or DMSO for 24 h. Then, enhancer activity was measured by luciferase assay using the Dual luciferase kit (Promega) according to the manufacturer’s instructions. Four replicates were included for each candidate enhancer.We randomly selected 58 H3K27ac-positive candidate enhancer elements after 20E treatment for luciferase assays. As enhancers were defined as H3K4me1 sites that were devoid of H3K4me3 modification, all of 58 enhancer elements were H3K4me1-positive and H3K4me3-negative. To test enhancer function in a reporter assay, we generated reporter constructs based on the pGL3-promotor backbone (Promega) by replacing the SV40 promoter sequence between Additional file 1. ChIP-seq enriched peaks of H3K4me3, H3K4me1, and H3K27ac after 20E treatment.Additional file 2. Genome-wide distribution of H3K4me1, H3K4me3, and H3K27ac modifications. a Pie charts showing the distribution of H3K4me-, H3K4me3-, and H3K27ac-enriched regions across the genome after 20E treatment. “Promoter-TSS” indicates the region − 1 kb to + 200 bp of the TSS. b Average ChIP-seq signal profiles for genes with different expression levels were generated for the histone modifications around the TSS after 20E treatment. Genes are divided into four categories according to their mRNA levels: no expression, low-level expression, medium-level expression, and high-level expression.Additional file 3. The overlap of each histone modification sites after 20E treatment.Additional file 4. Putative DREs (enhancers) and promoters were defined through modification-based prediction.Additional file 5.a The distribution of DREs. b IGV genome browser tracks of the FTZ-F1 locus with data for indicated histone modifications and RNA-seq. “+” and “−” indicate the presence and absence of 20E treatment, respectively. c GO-based gene functions for genes specifically associated with different types of enhancers. Red bars display functions based on nearest genes to H3K27ac-positive enhancers.Additional file 6. The primers and histone modification status of 58 candidate enhancer elements were listed. The results of luciferase assays of candidate enhancers were also listed in the last few columns.Additional file 7. ChIP-seq enriched peaks of EcR after 20E treatment.Additional file 8. The list of EcR peaks containing EcRE motif.Additional file 9. Motif analysis of EcR-enriched regions by MEME-ChIP and RSAT. a Motifs were enriched from EcR-binding sites of the arginine methyltransferase gene. b Average expression of the nearest genes of EcR. “EcR + EcRE” indicates EcR-peak regions enriched for the EcRE motif. “EcR − EcRE” indicated EcR-peak regions not enriched for EcRE motif. c Motifs enriched in distal EcR enhancer elements.Additional file 10. RPKM values for RNA-seq in BmE cells after treated with 20E or DMSO.


Dot1l in the murine telencephalon leads to cortical layering defects, indicating DOT1L activity and chromatin methylation at H3K79 impact on the cell cycle, and influence transcriptional programs conferring upper layer identity in early progenitors. Specifically, DOT1L prevents premature differentiation by increasing expression of genes that regulate asymmetric cell division . Loss of DOT1L results in reduced numbers of progenitors expressing genes including SoxB1 gene family members. Loss of DOT1L also leads to altered cortical distribution of deep layer neurons that express either TBR1, CTIP2 or SOX5, and less activation of transcriptional programs that are characteristic for upper layer neurons . Data from three different mouse models suggest that DOT1L balances transcriptional programs necessary for proper neuronal composition and distribution in the six cortical layers. Furthermore, because loss of DOT1L in the pre-neurogenic phase of development impairs specifically generation of SATB2-expressing upper layer neurons, our data suggest that DOT1L primes upper layer identity in cortical progenitors.Cortical development is controlled by transcriptional programs, which are orchestrated by transcription factors. Yet, stable inheritance of spatio-temporal activity of factors influencing cell fate and localization in different layers is only partly understood. Here we find that deletion of  Sox) family members, namely SOX2, SOX4, SOX5 and SOX11 . A growing body of data describes DOT1L function in different cell types, where it affects cell proliferation as well as other properties. But studies that aim to reveal the physiological roles of DOT1L rarely report on mechanisms or target genes that cause the reported phenotypes. Therefore, we aimed to address the function of DOT1L in balancing proliferation and differentiation during CNS development, where in vivo target genes are also unknown.Here, we report that activity of the Disruptor of telomeric silencing-like 1 (DOT1L) might prime UL neuronal identity already at the beginning of the neurogenic phase. DOT1L is a histone methyltransferase that mediates specifically histone H3 lysine 79 (H3K79) mono- (me1), di- (me2) and trimethylation (me3), although other enzymes may exist with the same function . DOT1L iThe data presented here show that DOT1L (i) prevents premature cell cycle exit of progenitors, at least in part by affecting asymmetric cell division, and (ii) supports transcriptional programs characteristic for UL cell fate during early cortical development. DOT1L thus primes progenitors for UL gene expression and cell fate before UL neuronal differentiation is thought to occur. Our data suggest that the H3K79me epigenetic modification might provide early-established cell fate information that is able to be transmitted to subsequent progenitor generations.Foxg1)-Cre/+ (Emx1)-Cre/+ (Nes)-CreER(T2)/R26R-(YFP)  were analyzed in comparison to littermates with Foxg1+/+ or Emx1+/+;Dot1lflox/+ (ctrl). For Nes-Cre line pregnant animals were injected with 200μg/g body weight tamoxifen (Tmx)  and 100 μg/g body weight progesterone . Non Tmx exposed animals were used as controls. Animal welfare committees of the University of Freiburg and local authorities approved all animal experiments . Genotyping was performed using primers listed in Forkhead box G1 (1)-Cre/+ , Empty s1)-Cre/+ , and Nes6R-(YFP)  animals ChIP-seq for H3K79me2 was performed from E12.5 entire forebrains, which were acutely dissociated into single cells and fixed as described . Data frISH, HE staining, and immunostaining of brain tissue and cultured cells was performed as previously described ,27. For IUE was carried out in C57BL/6  time-pregnant mice as previously described . BrieflyRNA-seq and ChIP-seq data were analyzed on the Galaxy platform . RNA-seqin vivo experiments each n is a different animal. For in vitro experiments each n was obtained from a different mESC differentiation. Exemplary data sets for cell numbers and qRTPCRs passed the D’Agostino-Pearson omnibus normality test. Cell numbers within a width of 200 μm of the cortex were normalized to the area in each bin (cell/mm2)  or Welch's t-test . For qRTPCRs data analysis, ΔCt values normalized to the house keeping gene Gapdh were compared using an unpaired, two-tailed Student's t-test  or Welch's t-test . For data presentation, ΔΔCt values are expressed as log2(fold change) and shown as mean ± SEM. These values were calculated from three technical replicates from each independent n. The error propagation formula was used to assess correct SEM values. For statistical analyses of γ-TUBULIN immunostainings, Fisher's exact test on a 2 × 2 contingency table with small sample size was used.Statistical comparisons were performed with GraphPad Prism 6 software. For ell/mm2) , and comDot1l is expressed in the progenitor zone and cortical plate between E11.5 and the adult stage  using a Foxg1-Cre driver line  studies suggested altered expression of Reelin (Reln) (Lhx2) in the knockout as compared to control mice -positive progenitors in the s Figure . Deep las Figure  were sigs Figure . In conts Figure , signifis Figure . TBR2-exs Figure . In situn (Reln)  (Supplemn (Reln) , and a trol mice .Dot1l-cKO reduced neural progenitor cells and promoted their premature differentiation into DL neurons, at the expense of UL neurons. To compare quantitated cell numbers in the microcephalic and control cortices, we divided the cortical plate into 10 bins and determined cell numbers in each bin. This approach allowed us to normalize to the variable height of the cortices in the different genotypes ) at E12.5 compared to control cells. At E14.5 the number of proliferating cells and the cycling fraction ) decreased in mutant compared to control animals  followed by genome-wide sequencing (ChIP-seq) at E12.5 and E14.5. Additionally, at E14.5 we performed ChIP-seq analysis of H3K4me3 that marks active transcription  differentially expressed (DE) genes upon Dot1l-cKO. We investigated the distribution of the H3K4me3 and H3K79me2 marks within the gene bodies  to the transcriptional end site (TES)) and adjacent ±4 kb of the 1976 DE genes  to identify specific DOT1L target genes that might be causing the observed phenotype and (2) to test this hypothesis.Foxg1 expression (caused by the Cre knockin) could lead to the transcriptional alterations observed in Dot1l-cKO , subventricular zone (SVZ), DL, UL, and layer 1 , Protein phosphatase 1 regulatory inhibitor subunit 1B (Ppp1r1b)) had lower H3K79me2 enrichment than UL expressed genes ). Taken together, ChIP-, RNA-seq, and qRTPCR data supported our hypothesis that direct DOT1L targets are most likely, but not exclusively, identified by high levels of H3K79me2 and decreased expression upon Dot1l-cKO. Among genes expressed by specific cell populations during cortical development, we identified Insulin like growth factor 2 mRNA binding protein 1 (Igf2bp1), Orthodenticle homeobox 1 (Otx1), Cyclin D1 (Ccnd1), Lhx2 and Nuclear factor I B (Nfib) as novel targets in progenitors, and Satb2 as well as Cux2 as UL targets. From transcripts expressed by DL neurons, only Sox5 classified as DOT1L target under our selection criteria. In addition, these data suggested to us that DOT1L might infer neuronal cell fate information because we identified target genes such as Satb2 and Cux2. These latter genes are expressed by late born neurons, but were marked with H3K79me2 already at a developmental stage dominated by proliferative events, i.e. E12.5.It is of note that the H3K79me2 pattern differed substantially between the selected genes Figure . We therSox1, Sox2, Sox4, Sox5, Sox6, Sox8, Sox10, Sox11, Transcription factor 7 like 1 (Tcf7l1), Tcf7l2, WD repeat and HMG-box DNA binding protein 1 (Wdhd1) and Thymocyte selection-associated HMG box (Tox1). With the exception of the oligodendrocyte-expressed Sox10  decreased the number of progenitors, and (iii) impacted on the generation and distribution of cortical plate neurons, depending on their respective subtype.This data supported the view that DOT1L and H3K79me2 (i) directly regulated HMG TF expression Dot1l deletion to identify further in vivo cortical DOT1L target genes. These analyses indicated enrichment for cell cycle-related genes (Ccnd1) and E2F transcription factor (E2f7), which are important for G1/S transition  immunostaining , and Centromere protein v (Cenpv) and j (Cenpj), which carried H3K79me2 modifications at E12.5 and E14.5, and Ankyrin repeat domain 6 (Ankrd6), which gained H3K79me2 at E14.5  into radial glial-like cells (RGLC) in vitro  were marked with H3K79me2 modification, and that their expression decreased upon loss of DOT1L , Crystallin mu (Crym), Synaptotagmin 9 (Syt9), Dkk3, Synuclein alpha (Scna), Adenylate cyclase 1 (Adcy1)) or UL , Cux1, Cux2)  were marked with H3K79me2 already at E12.5  at E12.5 to overexpress DOT1L, harvested brains at E14.5 and E16.5, and visualized electroporated cells by expression of RFP, which was co-electroporated. We quantified the total number of RFP-positive cells and determined the percentage of RFP-positive cells that co-expressed either PAX6, TBR2, TBR1, CTIP2 or SATB2. Quantitation of the fraction of double positive cells was restricted to the main expression domains of the respective marker genes. We did not observe alterations in E14.5 brains that overexpress DOT1L compared to the empty vector control electroporation  E9.5, E10.5, and E11.5, (ii) E11.5, E12.5, and E13.5 and (iii) E13.5, E14.5, and E15.5 to interfere with DOT1L expression during three different phases of cortical neurogenesis   established early in development, (ii) transmitted to future generations of progenitors, and (iii) kept at basal levels until full expression of a respective gene is needed. Thus, epigenetic modifications are important mechanisms to activate specific transcriptional programs in ESCs ,52, and  reports ,53,54, bsubunit) . In our subunit) ,59,60. Tin vivo target genes in mammalian cells are rare , the cKO at later stages gave slightly different patterns in the genesis of progenitors and neurons. Nes-Cre induced cKO between E11.5 and E13.5 did not result in different numbers of progenitors. It is tempting to speculate that progenitors are permissive toward loss of DOT1L at this stage. However, as we also observed increasing cell numbers of TBR1-, CTIP2-, and SATB2-neurons it seems also likely that cells enter the cell cycle in higher numbers and that loss of DOT1L accounts for a facilitated exit for differentiation into DL and UL subtypes in these stages. This view is supported by the altered cell cycle in Foxg1-Cre cKO mice (Figure Dot1l-cKO in this later developmental stage. However, as the distribution of SATB2-neurons significantly changed in the cortical plate upon DOT1L-deficiency in late neurogenesis, we conclude that DOT1L does not only function to balance the progenitor pool and to prime specific neuronal identity, but that it is also necessary for proper neuronal distribution in the developing cortex.Importantly, deletion of DOT1L in the three mouse models and at different developmental time points revealed that this enzyme has probably multiple functions during development. Compared to the loss of DOT1L in the pre-neurogenic phase (e Figure –G, in whDot1l-cKO vs ctrl: GSE95832, RNA-seq Foxg1-heterozygote vs ctrl: GSE95833. Following deep sequencing data have been deposited in NCBI BioProject: H3K79me2 E14.5: PRJNA282071, H3K4me3 E14.5: PRJNA287208.Following deep sequencing data have been deposited in GEO: H3K79me2 E12.5: GSE95831, RNA-seq Supplementary DataClick here for additional data file.


Supplemental Digital Content is available in the text. Vascular inflammation underlies cardiovascular disease. Vascular smooth muscle cells (VSMCs) upregulate selective genes, including MMPs  and proinflammatory cytokines upon local inflammation, which directly contribute to vascular disease and adverse clinical outcome. Identification of factors controlling VSMC responses to inflammation is therefore of considerable therapeutic importance. Here, we determine the role of Histone H3 lysine 9 di-methylation (H3K9me2), a repressive epigenetic mark that is reduced in atherosclerotic lesions, in regulating the VSMC inflammatory response.MMP3, MMP9, MMP12, and IL6, in mouse and human VSMCs. Inhibition of G9A/GLP (G9A-like protein), the primary enzymes responsible for H3K9me2, significantly potentiated inflammation-induced gene induction in vitro and in vivo without altering NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and MAPK (mitogen-activated protein kinase) signaling. Rather, reduced G9A/GLP activity enhanced inflammation-induced binding of transcription factors NFκB-p65 and cJUN to H3K9me2 target gene promoters MMP3 and IL6. Taken together, these results suggest that promoter-associated H3K9me2 directly attenuates the induction of target genes in response to inflammation in human VSMCs.We used VSMC-lineage tracing to reveal reduced H3K9me2 levels in VSMCs of arteries after injury and in atherosclerotic lesions compared with control vessels. Intriguingly, chromatin immunoprecipitation showed H3K9me2 enrichment at a subset of inflammation-responsive gene promoters, including This study implicates H3K9me2 in regulating the proinflammatory VSMC phenotype. Our findings suggest that reduced H3K9me2 in disease enhance binding of NFκB and AP-1 (activator protein-1) transcription factors at specific inflammation-responsive genes to augment proinflammatory stimuli in VSMC. Therefore, H3K9me2-regulation could be targeted clinically to limit expression of MMPs and IL6, which are induced in vascular disease. The repressive epigenetic mark Histone H3 Lysine 9 Dimethylation (H3K9me2) is reduced in vascular smooth muscle cells on inflammation in cardiovascular disease models.Loss of H3K9me2 in human and mouse vascular smooth muscle cells exacerbates inflammation-induced upregulation of disease-associated genes in vitro and in vivo, with functional consequences for matrix degradation and phenotypic switching.H3K9me2 inhibits binding of NFκB (nuclear factor kappa-light-chain-enhancer of activated B cell) and AP-1 (activator protein-1) transcription factors to their cognate sequences within MMP  and cytokine gene promoters.2199See accompanying editorial on page 2 Inflammation-responsive gene expression is controlled by the NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) and AP-1 (activator protein 1) transcription factors, and dysregulation of NFκB and MAPK (mitogen-activated protein kinase)/AP-1 signaling is linked to the initiation and progression of vascular dysfunction.3 Indeed, directly reducing inflammation with Canakinumab, an IL1-β neutralizing antibody, significantly lowered cardiovascular events compared with placebo in patients4 although not in mice.5 Identification of mechanisms that regulate inflammation-associated changes in VSMC gene expression is therefore of considerable therapeutic importance.Vascular smooth muscle cell (VSMC) accumulation is a hallmark of cardiovascular disease (CVD), including atherosclerosis and vascular remodeling after injury. In response to proinflammatory stimuli, VSMCs downregulate contractile proteins and upregulate selective gene sets, including matrix metalloproteinases, and proinflammatory cytokines , which directly contribute to the progression of vascular disease.7 For example, animal atherosclerosis studies implicate MMP12 in plaque development and instability.9 MMP3 and MMP9 promote VSMC migration and neointima formation after carotid ligation in mice.10 In addition, increased MMP3, MMP9, and MMP12 activity is observed in vulnerable compared with stable human atherosclerotic lesions.14 The proinflammatory cytokine IL-6 also plays an important role in vascular disease progression. IL-6 attenuates VSMC contractility while promoting VSMC migration, proliferation, and vascular calcification.17 Furthermore, elevated serum IL-6 is associated with greater CVD risk.20MMPs  comprise a family of proteases that degrade extracellular matrix components and mediate many structural changes associated with vascular disease.21 Decreased levels of histone H3 lysine 9 di-methylation (H3K9me2) and histone H3 lysine 27 tri-methylation (H3K27me3) are observed in VSMCs derived from human atherosclerotic plaques compared with control arteries.22 However, how these mechanisms control inflammation-associated changes in VSMC gene expression are unclear. Interestingly, H3K9me2, a repressive mark associated with facultative heterochromatin23 and directly regulating the expression of inducible genes in other cells,28 affects the growth rate, migration, and contractility of pulmonary and airway smooth muscle cells.30 Yet, the functional importance and target genes of H3K9me2 in VSMCs remain largely unknown.Gene expression is regulated by epigenetic pathways that modify histone proteins, resulting in either increased or reduced accessibility to the transcription machinery.MMP3, MMP9, MMP12, and IL6, which are strongly associated with CVD.14 Importantly, we show that H3K9me2 attenuates inflammation-associated upregulation of target genes by inhibiting NFκB/p65 and/or AP-1/cJUN transcription factor binding. These findings identify functional consequences of loss of H3K9me2 in vascular disease and suggest H3K9me2 as an important mechanism to prevent spurious induction of a proinflammatory state in VSMCs.We demonstrate that global levels of H3K9me2 in VSMCs are reduced in atherosclerosis and in arteries undergoing injury-induced remodeling, concomitant with increased inflammation. H3K9me2 is deposited at a subset of inflammation-responsive VSMC genes, including online-only Data Supplement.The chromatin immunoprecipitation (ChIP)-seq data have been made publicly available at the Gene Expression Omnibus with the accession number GSE131212. All other supporting data are available within the article and in the Apoe−/−) mice on a C57Bl/6 background were purchased from Charles River. Myh11-CreERt2 (Y-linked),31Rosa26-Confetti, Rosa26-EYFP (enhanced yellow fluorescent protein), and Apoe−/− mice have been described previously.32 MYH11 is a marker of differentiated smooth muscle cells and the Myh11-CreERt2 transgene used here has been extensively tested for VSMC-specific expression in major arteries.36 Experimental animals  received 10 intraperitoneal injections of 1 mg/mL tamoxifen between 4 and 8 weeks of age for lineage labeling. To inhibit G9A/GLP (G9A-like protein), lineage-labeled Myh11-CreERt2/Rosa26-EYFP+ males were treated with A366 delivered by osmotic pumps (30 mg/kg per day in 98:2 PEG 400/polysorbate 80), or vehicle alone, and either analyzed directly or subjected to ligation of the left common carotid artery as described previously.34 Surgery and tissue processing are described in the Methods in the online-only Data Supplement.All experiments were approved by the United Kingdom Home Office (PPL70/7565 and P452C9545) and the local ethics committee. Wild-type and Apolipoprotein E-null  was used to generate Illumina-compatible sequencing libraries from 100 pg to 2 ng of DNA from 2 independent H3K9me2 ChIP experiments and associated input. Libraries were sequenced (Illumina NextSeq) using paired-end 75 bp reads. ChIP-seq reads were trimmed using Cutadapt v1.9,37 aligned to the mouse GRCm38 genome using Bowtie2 v2.2,38 and reads per gene promoter (within ±1 kb of the transcription start sites) quantified using SeqMonk v1.4 (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk). Genes showing fewer than 20 read counts in the input samples were removed from further analysis and the ratio of H3K9me2/input signal was computed. Of genes with H3K9me2/input ratios in the top 25th percentile, 63 genes were associated with arteriosclerosis according to the Cardiovascular Disease Portal39 .ChIP was performed as described in the Methods in the 40 from patients undergoing aortic valve replacement with ethics committee approval and used at passage 6 to 15. Primary mouse aortic VSMCs were derived from 8- to 12-week-old wild-type C57Bl/6 males and used at passage 4. VSMCs were treated with UNC0638 , small interfering RNA targeting G9A , control small interfering RNA , SP600125 , human recombinant IL-1α , and human recombinant TNF-α (tumor necrosis factor-α), 90 ng/mL, Peprotech as indicated and analyzed as described in the Methods in the online-only Data Supplement.Human aortic VSMCs (hVSMCs) were isolated as describedU test, Kruskal-Wallis 1-way ANOVA with Dunn test to compare specific sample pairs, 1-sided exact Wilcoxon rank-sum tests or a linear model (described in the online-only Data Supplement) with P<0.05 as the threshold for considering results to be statistically significant.Data are shown as mean±SEM, unless otherwise indicated. The number of animals per group and number of biologically independent experiments are indicated in Figure legends (at least 3). Independent in vitro experiment were done using isolates derived from different individuals for hVSMCs and independent primary cultures for murine VSMCs. Data were analyzed using Mann-Whitney Myh11-CreERt2+ mice31 with recombination reporter alleles (Rosa26-Confetti32 or Rosa26-EYFP33). Tamoxifen treatment of these animals induces VSMC-specific recombination and stable fluorescent protein expression in 70% to 95% of VSMCs in Confetti34 and 40% to 60% in EYFP reporter mice.35 Importantly, the fluorescent lineage reporters are stably expressed, independent of the expression status of the Myh11-CreERt2 transgene, in medial VSMCs and in VSMC-derived neo-intimal and plaque cells.34VSMCs downregulate lineage markers in atherosclerosis and after injury, making specific quantification of H3K9me2 in VSMCs difficult. We therefore used genetic lineage tracing to definitively identify VSMCs by crossing Myh11-CreERt2/Rosa26-Confetti mice with Apoe−/− mice to assess H3K9me2 levels in atherosclerosis. Male mice received tamoxifen and were then fed an atherosclerosis-inducing high-fat diet  or a standard chow diet. Immunohistochemistry in carotid artery sections of animals after 3 to 4 months of high-fat diet revealed significantly lower H3K9me2 levels in Confetti+ cells within the media, core, and fibrous cap in atherosclerotic arteries compared with cells in control vessels , suggesting that H3K9me2 may regulate the inflammatory response of VSMCs.To assess whether H3K9me2 regulates early events in the VSMC inflammatory response, we performed ligation of the left common carotid artery, which elicits reproducible vessel remodeling concomitant with changes in VSMC gene expression, including induction of IL6 and MMPs.online-only Data Supplement), which is expected for a repressive epigenetic mark. We found that among the genes with high H3K9me2 levels within ±1kb of their transcription start site were 63 genes listed as arteriosclerosis-associated on the Cardiovascular Disease Portal39 (Table I in the online-only Data Supplement), including several inflammation-responsive MMPs, such as Mmp3, Mmp9, and Mmp12. ChIP-qPCR using cultured primary murine VSMCs confirmed abundant H3K9me2 levels at Mmp3, Mmp9, and Mmp12 as well as the positive control locus Magea2 compared with the negative control, Actb , Mmp9 (1.7-fold), and Mmp12 (7.1-fold)  compared with VSMCs treated with IL-1α alone  were administered A366, a G9A/GLP inhibitor that displays similar specificity and efficacy but improved pharmacokinetics compared with UNC063844. Two weeks of A366 treatment resulted in reproducible reduction of global H3K9me2 levels in VSMCs as revealed by immunostaining , endothelial cells (Cd31), or bone marrow-derived cells (Cd45), beyond background . Importantly, A366 did not affect expression of H3K9me2 target genes in uninjured vessels, the response of Ccl2 to ligation-injury, or the reduction of contractile VSMC gene expression after ligation . Together, these results suggest that H3K9me2 attenuates induction of inflammation-responsive H3K9me2 target genes in VSMCs in vivo and inhibits VSMC phenotypic switching to a more proinflammatory state.To assess the specific effect of H3K9me2 on injury-induced gene regulation in VSMCs, we performed reverse transcription with quantitative polymerase chain reaction on lineage-labeled, EYFP6 Figure E, correl6 Figure D. Interes Figure E. We con cells Cd5, beyondMMP3, MMP9, and MMP12 gene promoters compared with the active ACTB control locus with and without IL-1α treatment was conserved in human . Similar to murine tissue VSMCs, IL6 was also a H3K9me2 target in hVSMCs, whereas this repressive epigenetic mark was not present at CCL2 and MMP2 promoters . Expression levels of the IL-1 receptor was modestly increased in IL-1α-treated hVSMCs but was not affected by UNC-mediated G9A/GLP inhibition . Similar to IL-1α, TNF-α-mediated induction of H3K9me2 target genes MMP3, MMP9, MMP12, and IL6 was also potentiated by UNC, whereas MMP2 or CCL2 expression was not affected . This suggests that UNC acts downstream of receptor binding and affects a factor that is common to TNF-α and IL-1α signaling such as NFκB and MAPK-mediated activation of the AP-1 transcription factor . Binding of NFκB and AP-1 transcription factors to a number of MMP and proinflammatory cytokine promoters, including MMP352, MMP951, MMP12,51 and IL653, has been reported as necessary to induce their expression.In addition to their role as histone methyltransferases, G9A/GLP have nonhistone targets,54 Compared with untreated control cells, p65 levels  were unchanged in hVSMCs treated with UNC±IL-1α. UNC treatment neither affected the nuclear-to-cytoplasmic ratio of p65 at basal conditions nor its nuclear translocation in response to IL-1α or TNF-α stimulation . These results suggest that potentiation of IL-1α/TNF-α-induced IL6 and MMP expression by UNC is not because of increased upstream NFκB signaling.To investigate whether G9A/GLP regulates inflammation-induced NFκB activity, we analyzed regulation of the p65 subunit, which contains the transactivation domain necessary for gene induction.57 Western blotting showed that neither timing nor levels of phosphorylated JNK , ERK (extracellular regulating kinase) 1/2, and p38 kinase (p38) were affected by UNC treatment before IL-1α stimulation , suggesting that G9A/GLP inhibition does not influence MAPK activation.Inflammatory cytokines induce phosphorylation of MAPKs at specific residues, which ultimately leads to phosphorylation of AP-1 subunits, such as cJUN.MMP3, IL6, and CCL2  and cJUN (AP-1) in hVSMCs treated with IL-1α and UNC. Compared with the ACTB negative control locus, p65 was enriched at the IL6 and CCL2 gene promoters in untreated cells. At the IL6 locus, p65 binding was increased by IL-1α stimulation, and this was significantly enhanced by UNC pretreatment , the primary MAPK responsible for cJUN phosphorylation at Ser63.60 Treatment with SP600125 significantly reduced IL-1α-induced p-cJUN levels  or total cJUN . Reduced levels of p-cJUN in SP600125-treated cells selectively affected MMP3 gene expression resulting in an attenuated induction by IL-1α, whereas IL-1α-induced IL6 and CCL2 expression was not affected by JNK inhibition  and AP-1 (cJUN) transcription factor binding and demonstrates that inflammation-induced expression of MMP3, IL6, and CCL2 are regulated via different mechanisms.Taken together, these findings suggest that H3K9me2 acts directly at the MMP3, MMP9, MMP12, and IL6, as direct H3K9me2 targets in VSMCs. We show that loss of H3K9me2 increases binding of inflammation-induced transcription factors and increases expression of these MMP and proinflammatory VSMC genes in response to inflammatory stimuli both in vitro and in vivo. This functional role of H3K9me2 suggests that this epigenetic mark acts to restrain inflammation-induced, CVD-associated gene expression in VSMCs. It is therefore tempting to speculate that the reduced H3K9me2 levels we observed in atherosclerosis and after injury is required to elicit VSMC inflammatory activation and vascular remodeling. Increasing H3K9me2 levels by stimulating G9A/GLP activity or inhibiting H3K9me2-specific demethylases  might therefore be a strategy for therapeutic targeting in CVD.We here demonstrate an evolutionary conserved role of H3K9me2 in regulating the proinflammatory VSMC phenotype associated with vascular disease and identify a subset of inflammation-responsive genes, including 22 Our analysis of lineage traced VSMCs supports the observation that αSMA-positive plaque cells have lower H3K9me2 levels61 and further indicates that this epigenetic mark is also reduced in VSMC-derived cells within the lesion core, that do not express contractile markers.The global loss of H3K9me2 in VSMCs from atherosclerotic lesions and in arteries undergoing remodeling after vascular injury is consistent with the reported H3K9me2 reduction in atherosclerotic plaques from patients compared with healthy human arteries.63 VSMCs from diabetic patients also display reduced levels of H3K9me2 compared with nondiabetic controls, and KDM3A is induced by high insulin levels.64 Therefore, dysregulation of H3K9me2 might contribute to diabetes-associated vascular complications.H3K9me2-mediated regulation of inflammation-induced genes in VSMCs might also explain the accelerated neointima formation following vascular injury previously detected in diabetic rats when H3K9me2 levels were reduced by overexpression of KDM3A, a H3K9me2-specific demethylase.MMP and other inflammatory gene promoters and their expression has also been reported in other cell types.27 For example, H3K9me2 depletion at the Mmp9 promoter correlates with elevated Mmp9 expression in retinal endothelial cells from diabetic rats compared with controls.27 Similarly, solar-stimulated UV radiation induces loss of H3K9me2 at the MMP3 promoter in primary human dermal fibroblasts, correlating with enhanced MMP3 mRNA levels.25 In addition, genetic ablation or pharmacological inhibition of G9A/GLP stimulates the expression of inflammatory genes in mouse embryonic fibroblasts.24 Similar to our findings in VSMCs, reduction of H3K9me2 levels in fibroblasts potentiates poly(I:C)-induced upregulation of inflammatory genes.24 Moreover, G9A-deficient flies are hypersensitive to RNA virus infection and succumb faster to infection than wild-type controls.28 These observations suggest that G9A/GLP represents an evolutionarily conserved mechanism to control genes involved in processes that require tight and dynamic regulation—such as the inflammatory response—in a variety of cell types.An inverse correlation between H3K9me2 occupancy at 65 We propose that the 2-fold increase in p-cJUN in IL-1α-stimulated cells after G9A/GLP inhibition could be because of increased phosphorylation efficiency of chromatin-associated cJUN, which potentiates transactivation.67 Alternatively, G9A/GLP might regulate an as-yet-unidentified cJUN phosphatase.66 Regardless, our data demonstrate that key inflammation-induced genes have distinct regulatory mechanisms in VSMCs. Non-H3K9me2 targets such as CCL2 display transcription factor binding at basal levels resulting in rapid induction upon stimulation. In contrast, H3K9me2 target gene induction is delayed in a context-specific manner, which could depend on both specific transcription factor usage and locus-specific chromatin regulation.We demonstrate increased binding of AP-1 and NFκB specifically at H3K9me2 target genes on reduction of H3K9me2 levels following G9A/GLP inhibition . In contrast, inhibition of G9A/GLP did not affect IL-1α/TNF-α-induced activation of upstream signaling, including p65 activation and phosphorylation of p38, JNK, and ERK1/2. MAPKs can phosphorylate substrates once bound to chromatin.MMPs and IL6. Differential transcriptional regulation of selective MMPs and other inflammation-induced genes could be leveraged for developing more specific drugs for future clinical use.In conclusion, our data implicate H3K9me2 as a functional epigenetic regulator of the VSMC inflammatory response. We propose that H3K9me2 acts to prevent spurious induction of CVD-associated proinflammatory genes, including We thank the Cambridge Statistics Clinic (Department of Statistics) for advise on statistical analysis, Mikhail Spivakov for advise on chromatin immunoprecipitation-seq data analysis, Gregory Strachan and the Wellcome Trust-Medical Research Council, Institute of Metabolic Science, Metabolic Research Laboratories, Imaging core (Wellcome Trust Major Award [208363/Z/17/Z]) for technical assistance, the Cambridge National Institute for Health Research Biomedical Research Centre Cell Phenotyping Hub for cell sorting and all members of the Jørgensen group for helpful discussions.J.L. Harman was funded by a British Heart Foundation (BHF) studentship (FS/15/38/31516); L. Dobnikar was funded by a BBSRC DTP studentship; J. Chappell was funded by the BHF Centre for Cardiovascular Research Excellence (RE/13/6/30180); B.G. Stokell was funded by a studentship from the Cantab Capital Institute for the Mathematics of Information. K. Foote was funded by the BHF PG/16/63/32307; A. Finigan and A. Uryga were funded by the BHF RG/13/14/30314. A.L. Taylor was funded by BHF studentship (FS/15/62/32032). M.R. Bennett and H.F. Jørgensen were supported by the BHF (CH/2000003), the Oxbridge BHF Centre for Regenerative Medicine , the BHF Centre for Cardiovascular Research Excellence (RE/13/6/30180), and the National Institute for Health Research Cambridge Biomedical Research Center.None.