Conserved Noncoding Sequences Research Articles

Heart enhancers with deeply conserved regulatory activity are established early in zebrafish development

During the phylotypic period, embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. Here we set out to identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period. We isolate early cardiac progenitor cells from zebrafish embryos and characterize 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlap with conserved non-coding elements (CNEs) that also map to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drive gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discover 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period.

Conserved Noncoding Sequences Boost ADR1 and SP1 Regulated Human Swiprosin-1 Promoter Activity

Swiprosin-1 is expressed in various types of cells or tissues of different species. To investigate the mechanisms underlying Swiprosin-1 expression pattern, we analyzed the promoter activity of 2-kilobase genomic sequences located at 5′ flanking region of the Swiprosin-1 gene. The −2000/+41 bp of 5′ flanking untranslated promoter region of Swiprosin-1 gene was constitutively transactivated without significant effect of PMA, A23187, or PMA/A23187 stimulation in Jurkat T cells. Further, we identified 5′ deletant of proximal promoter region (−100/+41 to −70/+41) plays a pivotal role in activating the Swiprosin-1 gene in Jurkat T cells. Our studies also verified that ADR1 and Sp1 transcription factors were located between −70 and -100 locus of 5′ flanking proximal promoter region, which is critical for the Swiprosin-1 promoter activity. ADR1 and Sp1 were shown to bind the regions of −82, −79, −76, −73 and −70 and; −79, −78 and −77, respectively, within the proximal promoter region of Swiprosin-1. Finally conserved noncoding sequences (CNS) -1, -2 and -3 were located between the exon 1 and exon 2 of Swiprosin-1 gene and synergistically transactivated the Swiprosin-1 promoter. In summary, Swiprosin-1 was constitutively expressed in Jurkat T cells by the coordinate action of ADR1 and SP1 transcription factors at the transcriptional level and CNS further boost the proximal region of Swiprosin-1 promoter activity. Our findings provide novel insights that the transcriptional regulation of Swiprosin-1 by targeting ADR1 and Sp1 binding sites may be helpful in exploring novel therapeutic strategies for advanced immune or other disorders.

Development and Functional Modulation of Regulatory T Cells by Transcription Factors and Epigenetics.

Regulatory T cells (Tregs) are essential for the maintenance of immune homeostasis. Studies of Treg are not only necessary for understanding the mechanism of immune homeostasis but also extremely useful for the development of treatments of various immune diseases. Forkhead box P3 (Foxp3) was identified as the master gene responsible for the immune-suppressing activity of Tregs. The promoter region and several intronic enhancers, designated conserved noncoding sequence (CNS) 0, 1, 2, and 3, at the Foxp3 gene locus have important roles in Foxp3 expression and Treg development. We demonstrated that transcription factors Nr4a and Smad2/3 are required for development of thymic Tregs and induced Tregs, respectively. In addition to transcription factors, Treg-specific DNA demethylation has been shown to be important for Treg stability. In particular, DNA demethylation of CNS2 was implicated in Treg stability, and members of the ten-eleven translocation family of demethylation factors were recently demonstrated to have important roles in 5'-C-phosphate-G-3' demethylation at CNS2. This article summarizes recent findings regarding the roles of transcription factors and epigenetic modifications in the differentiation, maintenance, and function of Tregs. This review will facilitate clinical application of Tregs to diseases in the field of ophthalmology, including uveitis and age-related macular degeneration.

Pairs of Adjacent Conserved Noncoding Elements Separated by Conserved Genomic Distances Act as Cis-Regulatory Units.

Comparative genomic studies have identified thousands of conserved noncoding elements (CNEs) in the mammalian genome, many of which have been reported to exert cis-regulatory activity. We analyzed ∼5,500 pairs of adjacent CNEs in the human genome and found that despite divergence at the nucleotide sequence level, the inter-CNE distances of the pairs are under strong evolutionary constraint, with inter-CNE sequences featuring significantly lower transposon densities than expected. Further, we show that different degrees of conservation of the inter-CNE distance are associated with distinct cis-regulatory functions at the CNEs. Specifically, the CNEs in pairs with conserved and mildly contracted inter-CNE sequences are the most likely to represent active or poised enhancers. In contrast, CNEs in pairs with extremely contracted or expanded inter-CNE sequences are associated with no cis-regulatory activity. Furthermore, we observed that functional CNEs in a pair have very similar epigenetic profiles, hinting at a functional relationship between them. Taken together, our results support the existence of epistatic interactions between adjacent CNEs that are distance-sensitive and disrupted by transposon insertions and deletions, and contribute to our understanding of the selective forces acting on cis-regulatory elements, which are crucial for elucidating the molecular mechanisms underlying adaptive evolution and human genetic diseases.

CNEFinder: finding conserved non-coding elements in genomes.

MotivationConserved non-coding elements (CNEs) represent an enigmatic class of genomic elements which, despite being extremely conserved across evolution, do not encode for proteins. Their functions are still largely unknown. Thus, there exists a need to systematically investigate their roles in genomes. Towards this direction, identifying sets of CNEs in a wide range of organisms is an important first step. Currently, there are no tools published in the literature for systematically identifying CNEs in genomes.ResultsWe fill this gap by presenting ; a tool for identifying CNEs between two given DNA sequences with user-defined criteria. The results presented here show the tool’s ability of identifying CNEs accurately and efficiently. is based on a k-mer technique for computing maximal exact matches. The tool thus does not require or compute whole-genome alignments or indexes, such as the suffix array or the Burrows Wheeler Transform (BWT), which makes it flexible to use on a wide scale.Availability and implementationFree software under the terms of the GNU GPL (https://github.com/lorrainea/CNEFinder).

Draft genome of Dugesia japonica provides insights into conserved regulatory elements of the brain restriction gene nou-darake in planarians

BackgroundPlanarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far.ResultsTo obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates.ConclusionThis draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp.

Strong TCR stimulation promotes the stabilization of Foxp3 expression in regulatory T cells induced in vitro through increasing the demethylation of Foxp3 CNS2

Foxp3 is the master transcriptional regulator of regulatory T cells (Tregs), and the stabilization of Foxp3 expression is regulated by the demethylation of conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Recent studies have shown that TCR stimulation is required for the demethylation of Foxp3 CNS2 during Treg development. However, the relationship between the strength of TCR stimulation and the demethylation of Foxp3 CNS2 remains unclear. To address this issue, we compared the frequency of demethylation of the Foxp3 CNS2 among in vitro-induced Tregs (iTreg) that had received a range of TCR stimulation during their development. We found that the frequency of demethylation of the Foxp3 CNS2 was increased with increased TCR stimulation strength, whereas CD28 stimulation had only a limited effect. Mechanistically, the binding of Tet2, a member of the TET family of enzymes involved in DNA demethylation, on the Foxp3 CNS2 was increased by strong TCR stimulation. Furthermore, compared with iTreg induced by weak TCR stimulation, iTreg induced by strong TCR stimulation maintained Foxp3 expression both in vitro and in vivo. These data indicate that the strength of TCR stimulation is a key factor for induction of the demethylation of Foxp3 CNS2 and the generation of stable Tregs.

Finding Genetic Regulators, Forecasting Function

Finding Genetic Regulators, Forecasting Function

Conserved Noncoding Elements Influence the Transposable Element Landscape in Drosophila.

Highly conserved noncoding elements (CNEs) constitute a significant proportion of the genomes of multicellular eukaryotes. The function of most CNEs remains elusive, but growing evidence indicates they are under some form of purifying selection. Noncoding regions in many species also harbor large numbers of transposable element (TE) insertions, which are typically lineage specific and depleted in exons because of their deleterious effects on gene function or expression. However, it is currently unknown whether the landscape of TE insertions in noncoding regions is random or influenced by purifying selection on CNEs. Here, we combine comparative and population genomic data in Drosophila melanogaster to show that the abundance of TE insertions in intronic and intergenic CNEs is reduced relative to random expectation, supporting the idea that selective constraints on CNEs eliminate a proportion of TE insertions in noncoding regions. However, we find no evidence for differences in the allele frequency spectra for polymorphic TE insertions in CNEs versus those in unconstrained spacer regions, suggesting that the distribution of fitness effects acting on observable TE insertions is similar across different functional compartments in noncoding DNA. Our results provide evidence that selective constraints on CNEs contribute to shaping the landscape of TE insertion in eukaryotic genomes, and provide further evidence that CNEs are indeed functionally constrained and not simply mutational cold spots.

Identification of a conserved multi-lineage Il9 enhancer

Abstract The immune system provides resistance to the myriad of pathogens in the environment, but can also respond inappropriately causing allergic inflammation and autoimmune disease. CD4+ T cells, which play a crucial role in adaptive immune system, can be divided into subsets based on their effector functions. T helper 9 (Th9) cells, derived by the IL-4/STAT6 and TGFβ signaling pathways, produce IL-9 as a hallmark cytokine. Through IL-9 production, Th9 cells protect against parasite infection but are also involved in allergic inflammation and autoimmune diseases. In this study, we demonstrate that a conserved noncoding sequence (CNS) located 25kb upstream of the Il9 transcription start site, termed Il9 CNS-25, is critical for regulating Il9 expression in Th cell subsets and IL-9 producing CD8+ T cells (Tc9). Th9 cells derived from Il9 CNS-25 mutant (Il9ΔCNS-25) mice produce significantly less IL-9. Il9 CNS-25 was bound by factors that promote Il9 gene expression including STATs, IRF4 and BATF, promoted chromatin modifications and transcription factor binding at the promoter, and mediated accessibility of the locus. Il9ΔCNS-25 mice showed attenuated airway inflammation compared to control mice. The Il9 CNS-25 region in mice is conserved with an IL9 CNS-18 region in the human genome. We deleted CNS-18 in primary human Th9 cells and observed diminished IL-9 production. Thus, we have demonstrated that the Il9 CNS-25/IL9 CNS-18 elements are respectively critical for Il9/IL9 gene expression in Th9 cells and other IL-9 producing T cell subsets.

Allergic TH2 Response Governed by B-Cell Lymphoma 6 Function in Naturally Occurring Memory Phenotype CD4+ T Cells.

Transcriptional repressor B-cell lymphoma 6 (Bcl6) appears to regulate TH2 immune responses in allergies, but its precise role is unclear. We previously reported that Bcl6 suppressed IL-4 production in naïve CD4+ T cell-derived memory TH2 cells. To investigate Bcl6 function in allergic responses in naturally occurring memory phenotype CD4+ T (MPT) cells and their derived TH2 (MPTH2) cells, Bcl6-manipulated mice, highly conserved intron enhancer (hcIE)-deficient mice, and reporter mice for conserved noncoding sequence 2 (CNS2) 3′ distal enhancer region were used to elucidate Bcl6 function in MPT cells. The molecular mechanisms of Bcl6-mediated TH2 cytokine gene regulation were elucidated using cellular and molecular approaches. Bcl6 function in MPT cells was determined using adoptive transfer to naïve mice, which were assessed for allergic airway inflammation. Bcl6 suppressed IL-4 production in MPT and MPTH2 cells by suppressing CNS2 enhancer activity. Bcl6 downregulated Il4 expression in MPTH2 cells, but not MPT cells, by suppressing hcIE activity. The inhibitory functions of Bcl6 in MPT and MPTH2 cells attenuated allergic responses. Bcl6 is a critical regulator of IL-4 production by MPT and MPTH2 cells in TH2 immune responses related to the pathogenesis of allergies.

Elevated O‐GlcNAc Exacerbates Pro‐Inflammatory Cytokine Secretion from CD4+ T cells

Chronic inflammation is a feature of obesity and enhances the risk of atherosclerosis, cancer, diabetes, and autoimmunity. Specifically, pro-inflammatory TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell function. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a sensor of a cell's nutritional status. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells from mice fed a high fat and cholesterol “Western” diet. Naïve CD4+ T cells from obese mice have elevated O-GlcNAc levels compared to cells from mice fed standard chow. When polarized to a TH17 lineage, cells from obese mice secrete more IL-17A, the eponymous TH17 cytokine. Importantly, when naïve CD4+ T cells from lean and obese mice are polarized to a TH17 lineage in the presence of Thiamet-G (TMG, an inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA)) the cells from lean mice secrete levels of IL-17 comparable to those from obese mice and IL-17 secretion was exacerbated in cells from obese mice. Transcript levels of IL-17 are similarly elevated in both lean and obese mice TH17 cells treated with TMG. RORγt (retinoic acid-related orphan receptor gamma) acts as the TH17 master transcription factor, orchestrating the TH17 differentiation program. Uniquely among the CD4+ effector T cell master transcription factors, RORγt is regulated by fatty acid ligands which promote or repress its activity. Acetyl CoA carboxylase 1 (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt. We discovered that ACC1 is O-GlcNAcylated. Furthermore, lipidomics analysis identified clear differences in the lipid environment of T cells isolated from lean and obese mice −/+ TMG. Thus, abnormally elevated O-GlcNAc levels may favor the generation of ligands that enhance RORγt activity at the IL-17 promoter. More directly implicating O-GlcNAc's regulation of RORγt, chromatin immunoprecipation of RORγt and O-GlcNAc transferase (OGT, the enzyme that modifies proteins with O-GlcNAc) at the IL-17 promoter and an enhancer, CNS2 (conserved noncoding sequence 2), is increased with TMG treatment. Collectively, our data suggest that elevated O-GlcNAc levels increase pro-inflammatory IL-17 secretion from TH17 cells through alteration of the lipidome and direct effects on the IL-17 promoter complex. Further study into the molecular mechanism of how O-GlcNAcylation promotes pro-inflammatory TH17 T cell function will provide insight into how nutritional excess worsens inflammation. Support or Funding Information This research was supported by NIH grant R01DK100595-01, NIH grant R01DK091277-03, COBRE P30GM122731, and a KUMC Institute of Reproductive Health and Regenerative Medicine pilot grant. MM is supported by a KUMC Biomedical Research Training Program grant. Elevated O-GlcNAc increases pro-inflammatory IL-17 cytokine secretion from TH17 cells This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Enhancer trapping nearby rps26 gene in zebrafish mediated by the Tol2 transposon and it's annotation

With the completion of large-scale genome sequencing of human beings and other organisms, understanding the expression of control elements on the genome has become an important research task in the post-genome era. The enhancer trapping technology is an effective method for identifying enhancer elements in the genome and understanding its mechanism for gene expression regulation. In this study, we selected the stable enhancer trapping line TK4 (head and trunk specific GFP expression), which is generated with the mediation of Tol2 transposon system, and analyzed the trapped enhancers with the techniques of Splinkerette PCR (sp-PCR), in situ hybridization and comparative genomics. We crossed F1 individuals of TK4 line with wild-type zebrafish, collected fertilized eggs, and then detected the expression pattern of green fluorescent protein reporter gene by fluorescence microscopy at six different developmental stages, 6 hpf (hour post fertilization), 24 hpf, 48 hpf, 3 dpf (day post fertilization), 4 dpf and 5 dpf . The zebrafish genome flank sequence near the insertion site of Tol2 transposon was cloned by sp-PCR, and the results revealed that the insertion located at the position 27749253 of chromosome 23, and the transgene inserted reversely inside the intron 1 of rps26 gene. Within the 100 kb region of the insertion site, totally, seven genes including arf3a, wnt10b, wnt1, rps26, IKZF4, dnajc22 and lmbr1l were identified. Comparative genomic analysis by VISTA program revealed that there were two potential enhancer elements in the downstream of rps26 gene, which were conserved non-coding sequence (CNS) 1 and CNS2. The results of in situ hybridization showed that two transcripts of rps26 gene were maternal expression, the expression of rps26-201 in zygote was earlier than that of rps26-001, and the GFP signal of TK4 line zebrafish was not detectable before 6hpf, the expression patterns of rps26 and GFP at the late stages display similarity, and also represent differences, which suggested that the expression of rps26 and GFP may be controlled by the same enhancer, and also by the different enhancer, and two potential enhancers (CNS1 and CNS2) may play a differential regulation roles on the spatial and temporal expression of nearby genes (including rps26). In this study, we successfully obtained two potential enhancers near rps26 gene for the first time, which laid a foundation for further study of the regulation mechanism between these two enhancers and nearby genes in the genome, and the combination technique used in this study also provides a reference for enhancer analysis.

Proliferation of Regulatory DNA Elements Derived from Transposable Elements in the Maize Genome.

Genomic regions free of nucleosomes, which are hypersensitive to DNase I digestion, are known as DNase I hypersensitive sites (DHSs) and frequently contain cis-regulatory DNA elements. To investigate their prevalence and characteristics in maize (Zea mays), we developed high-resolution genome-wide DHS maps using a modified DNase-seq technique. Maize DHSs exhibit depletion of nucleosomes and low levels of DNA methylation and are enriched with conserved noncoding sequences (CNSs). We developed a protoplast-based transient transformation assay to assess the potential gene expression enhancer and/or promoter functions associated with DHSs, which showed that more than 80% of DHSs overlapping with CNSs showed an enhancer function. Strikingly, nearly 25% of maize DHSs were derived from transposable elements (TEs), including both class I and class II transposons. Interestingly, TE-derived DHSs (teDHSs) homologous to retrotransposons were enriched with sequences related to the intrinsic cis-regulatory elements within the long terminal repeats of retrotransposons. We demonstrate that more than 80% of teDHSs can drive transcription of a reporter gene in protoplast assays. These results reveal the widespread occurrence of TE-derived cis-regulatory sequences and suggest that teDHSs play a major role in transcriptional regulation in maize.

Conserved noncoding sequences conserve biological networks and influence genome evolution.

Comparative genomics approaches have identified numerous conserved cis-regulatory sequences near genes in plant genomes. Despite the identification of these conserved noncoding sequences (CNSs), our knowledge of their functional importance and selection remains limited. Here, we used a combination of DNA methylome analysis, microarray expression analyses, and functional annotation to study these sequences in the model tree Populus trichocarpa. Methylation in CG contexts and non-CG contexts was lower in CNSs, particularly CNSs in the 5'-upstream regions of genes, compared with other sites in the genome. We observed that CNSs are enriched in genes with transcription and binding functions, and this also associated with syntenic genes and those from whole-genome duplications, suggesting that cis-regulatory sequences play a key role in genome evolution. We detected a significant positive correlation between CNS number and protein interactions, suggesting that CNSs may have roles in the evolution and maintenance of biological networks. The divergence of CNSs indicates that duplication-degeneration-complementation drives the subfunctionalization of a proportion of duplicated genes from whole-genome duplication. Furthermore, population genomics confirmed that most CNSs are under strong purifying selection and only a small subset of CNSs shows evidence of adaptive evolution. These findings provide a foundation for future studies exploring these key genomic features in the maintenance of biological networks, local adaptation, and transcription.

Single-Base Resolution Map of Evolutionary Constraints and Annotation of Conserved Elements across Major Grass Genomes.

Conserved noncoding sequences (CNSs) are evolutionarily conserved DNA sequences that do not encode proteins but may have potential regulatory roles in gene expression. CNS in crop genomes could be linked to many important agronomic traits and ecological adaptations. Compared with the relatively mature exon annotation protocols, efficient methods are lacking to predict the location of noncoding sequences in the plant genomes. We implemented a computational pipeline that is tailored to the comparisons of plant genomes, yielding a large number of conserved sequences using rice genome as the reference. In this study, we used 17 published grass genomes, along with five monocot genomes as well as the basal angiosperm genome of Amborella trichopoda. Genome alignments among these genomes suggest that at least 12.05% of the rice genome appears to be evolving under constraints in the Poaceae lineage, with close to half of the evolutionarily constrained sequences located outside protein-coding regions. We found evidence for purifying selection acting on the conserved sequences by analyzing segregating SNPs within the rice population. Furthermore, we found that known functional motifs were significantly enriched within CNS, with many motifs associated with the preferred binding of ubiquitous transcription factors. The conserved elements that we have curated are accessible through our public database and the JBrowse server. In-depth functional annotations and evolutionary dynamics of the identified conserved sequences provide a solid foundation for studying gene regulation, genome evolution, as well as to inform gene isolation for cereal biologists.

Vitamin C Stabilizes Foxp3 Expression in Induced Treg Cells By Targeted DNA Demethylation and Prevents Murine Model of Acute Graft Versus Host Disease

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess of immune responses following allogeneic hematopoietic stem cell transplantation. Although antigen-specific in vitro- expanded Tregs (iTregs) have long been considered as a promising therapeutic agent against allo-immune reactions such as graft versus host disease (GVHD), accumulating evidences have suggested that iTregs easily lose their characteristics with quick cessation of Foxp3 expression , a lineage specifying transcription factor of Tregs, compared with in vivo -generated natural Tregs (nTregs). It has been revealed that insufficient demethylation of the CpG islands in conserved noncoding sequence 2 (CNS2) region of the Foxp3 locus mainly explains such instability of Foxp3 expression in iTregs.In order to overcome this drawback, we investigated the optimum way to generate stable iTregs for the prevention of a murine model of acute GVHD. We created a major-MHC mismatched GVHD model by transferring CD45.1 (H-2Kb) bone marrow (BM) cells together with effector T cells (CD4+25- and CD8 cells) into lethally irradiated (8Gy/body) 8-12-week old BALB/C (H-2Kd) recipients. In this model, recipient mice usually die within 7-10 days after bone marrow transplant (BMT) due to severe acute GVHD. Alloantigen-specific iTregs were generated by co-culturing naive T cells from human CD2 Foxp3 reporter mice (C57BL6/J background, H-2Kb) with BALB/C-derived antigen presenting cells in the presence of TGF-β, IL-2 and retinoic acid. Alloantigen-specific iTregs were then harvested 6-7 days after co-culture, by FACS-sorting the CD4+Foxp3+ population. Subsequently, sorted iTregs (106 cells) were transferred intravenously together with effector T cells (106 cells) and BM cells into recipients. We tested a panel of pharmacological agents and gene transduction during co-culture for their effects on iTreg stability, with FACS-based evaluation on day 7 after BMT. Among a panel of agents and gene modification tested, we found that only vitamin C-treated iTregs effectively improved Foxp3 maintenance compared with untreated iTregs (90%, vitamin C-treated iTregs vs 40%, untreated iTregs) in the GVHD model mice. As reported, vitamin C facilitated DNA demethylation of the Foxp3 CNS2 in iTregs in a Tet DNA demethylase-dependent manner. Bisulfite sequencing revealed a significant acceleration of CpG demethylation at the Foxp3 CNS2 by vitamin C, and the extent of demethylation achieved with vitamin C treatment reached to an equivalent level to those seen in nTregs. Furthermore, vitamin C mediated demethylation was extended to other Treg cell-specific regulatory lesions such as Tnfrsf18, Ikzf4, Ctla4 . On the other hand, untreated iTregs remain methylated at these loci to the same degree as naïve T cells (p<0.05) . At the same time, interestingly, production of inflammatory cytokines such as IFNγ and IL-21 that was observed in untreated iTregs which lost Foxp3 expression (ex- Foxp3 iTreg) was not observed in vitamin C-treated counterparts. Additionally, we further evaluated the benefits of vitamin C treatment, by investigating effects of another well-known demethylating agent, 5-aza-2'-deoxytidine (5-aza-dC). As a result, 5-aza-dC did not only induce DNA demethylation of Foxp3 CNS2, but also of the CNS1 enhancer region of the inflammatory cytokine IFNγ locus, even in iTreg conditioning culture. This nonspecific demethylation was not observed in vitamin C-treated iTregs (p<0.01). Finally , adoptive transfer of vitamin C-treated iTregs ameliorated GVHD in mice. Vitamin C-treated iTreg prolonged survival at BMT day 45 compared with untreated iTreg (100%, N=8, vitamin C treated iTreg group, vs 44.4%, untreated iTreg group, N=9, p<0.01). Amelioration of acute GVHD by vitamin C-treated iTreg was also confirmed by colon length on day 7 after BMT (p<0.05) and pathological findings of colon and small intestine.In summary, vitamin C stabilized Foxp3 expression with specific demethylation of Foxp3 CNS2 as well as Treg-associated genes in antigen-specific iTregs, preventing them from conversion into inflammatory ex- Foxp3 iTregs, thus ameliorating the pathogenesis of a murine model of GVHD. Our findings strongly suggest the potential of clinical application of Treg therapy combined with vitamin C treatment, for GVHD and autoimmune diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Conserved non-coding elements: developmental gene regulation meets genome organization.

Comparative genomics has revealed a class of non-protein-coding genomic sequences that display an extraordinary degree of conservation between two or more organisms, regularly exceeding that found within protein-coding exons. These elements, collectively referred to as conserved non-coding elements (CNEs), are non-randomly distributed across chromosomes and tend to cluster in the vicinity of genes with regulatory roles in multicellular development and differentiation. CNEs are organized into functional ensembles called genomic regulatory blocks–dense clusters of elements that collectively coordinate the expression of shared target genes, and whose span in many cases coincides with topologically associated domains. CNEs display sequence properties that set them apart from other sequences under constraint, and have recently been proposed as useful markers for the reconstruction of the evolutionary history of organisms. Disruption of several of these elements is known to contribute to diseases linked with development, and cancer. The emergence, evolutionary dynamics and functions of CNEs still remain poorly understood, and new approaches are required to enable comprehensive CNE identification and characterization. Here, we review current knowledge and identify challenges that need to be tackled to resolve the impasse in understanding extreme non-coding conservation.

Generation of allo-antigen-specific induced Treg stabilized by vitamin C treatment and its application for prevention of acute graft versus host disease model.

Antigen-specific regulatory T cells (Tregs) possess the potential to reduce excess immune responses in autoimmune diseases, allergy, rejection after organ transplantation and graft-versus-host disease (GVHD) following hematopoietic stem cell transplantation. Although in vitro-expanded antigen-specific induced Tregs (iTregs) have been considered to be a promising therapeutic agent against such excessive immune reactions, the instability of iTregs after transfer is a fundamental problem in their clinical application. In this study, we searched for the optimal way to generate stable iTregs for the prevention of the murine GVHD model, in which conventional iTregs are reported to be inefficient. Allo-antigen-specific iTregs were generated by co-culturing naive T cells with allogenic dendritic cells in the presence of TGF-β and retinoic acid. By examining various agents and genes, we found that vitamin C stabilized Foxp3 expression most effectively in adoptively transferred iTregs under a GVHD environment. Vitamin C treatment caused active DNA demethylation specifically on the conserved non-coding sequence 2 (CNS2) enhancer of the Foxp3 gene locus in allo-antigen-specific iTregs and reduced iTreg conversion into pathogenic exFoxp3 cells. Vitamin C-treated iTregs suppressed GVHD symptoms more efficiently than untreated iTregs. Vitamin C also facilitated induction of a FOXP3high iTreg population from human naive T cells, which was very stable even in the presence of IL-6 in vitro. The treatment of vitamin C for iTreg promises innovative clinical application for adoptive Treg immunotherapy.

Topologically associating domains are ancient features that coincide with Metazoan clusters of extreme noncoding conservation

Developmental genes in metazoan genomes are surrounded by dense clusters of conserved noncoding elements (CNEs). CNEs exhibit unexplained extreme levels of sequence conservation, with many acting as developmental long-range enhancers. Clusters of CNEs define the span of regulatory inputs for many important developmental regulators and have been described previously as genomic regulatory blocks (GRBs). Their function and distribution around important regulatory genes raises the question of how they relate to 3D conformation of these loci. Here, we show that clusters of CNEs strongly coincide with topological organisation, predicting the boundaries of hundreds of topologically associating domains (TADs) in human and Drosophila. The set of TADs that are associated with high levels of noncoding conservation exhibit distinct properties compared to TADs devoid of extreme noncoding conservation. The close correspondence between extreme noncoding conservation and TADs suggests that these TADs are ancient, revealing a regulatory architecture conserved over hundreds of millions of years.