Conserved Noncoding Sequences Research Articles

Two histone/protein acetyltransferases, CBP and p300, are indispensable for Foxp3+ T-regulatory cell development and function.

T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.

Human cytomegalovirus drives epigenetic imprinting of the IFNG locus in NKG2Chi natural killer cells.

Memory type 1 T helper (TH1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8+ T cells or TH1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2Chi NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

6-Methoxyflavone inhibits NFAT translocation into the nucleus and suppresses T cell activation.

NFAT plays a crucial role in the immune system by regulating the transcription of inducible genes during immune responses. In T cells, NFAT proteins govern various cellular events related to T cell development, activation, tolerance induction, and differentiation. We previously reported the NFAT1-dependent enhancer activity of conserved noncoding sequence (CNS)-9, a distal cis-acting element, in the regulation of IL-10 transcription in T cells. In this study, we developed a T cell-based reporter system to identify compounds that modulate the regulatory activity of CNS-9. Among the identified candidates, 6-methoxyflavone (6-MF) significantly inhibited the enhancer activity of CNS-9, thereby reducing IL-10 expression in T cells without affecting cell viability. 6-MF also downregulated the transcription of NFAT1 target genes such as IL-4, IL-13, and IFN-γ. Treatment of 6-MF inhibited the translocation of NFAT1 into the nucleus, which consequently interrupted NFAT1 binding to the target loci, without affecting the expression or dephosphorylation of NFAT1. Treatment of 6-MF to CD4(+) T cells or B cells isolated from mice with atopic dermatitis significantly reduced disease-associated cytokine production, as well as the levels of IgE. In addition, oral administration of 6-MF to atopic dermatitis mice ameliorated disease symptoms by reducing serum IgE levels and infiltrating lymphocytes. Conclusively, our results suggest that 6-MF can be a potential candidate for the development of an effective immunomodulator via the suppression of NFAT-mediated T cell activation.

Lineage-specific conserved noncoding sequences of plant genomes: their possible role in nucleosome positioning.

Many studies on conserved noncoding sequences (CNSs) have found that CNSs are enriched significantly in regulatory sequence elements. We conducted whole-genome analysis on plant CNSs to identify lineage-specific CNSs in eudicots, monocots, angiosperms, and vascular plants based on the premise that lineage-specific CNSs define lineage-specific characters and functions in groups of organisms. We identified 27 eudicot, 204 monocot, 6,536 grass, 19 angiosperm, and 2 vascular plant lineage-specific CNSs (lengths range from 16 to 1,517 bp) that presumably originated in their respective common ancestors. A stronger constraint on the CNSs located in the untranslated regions was observed. The CNSs were often flanked by genes involved in transcription regulation. A drop of A+T content near the border of CNSs was observed and CNS regions showed a higher nucleosome occupancy probability. These CNSs are candidate regulatory elements, which are expected to define lineage-specific features of various plant groups.

Function of a Foxp3 cis-Element in Protecting Regulatory T Cell Identity

The homeostasis of multicellular organisms requires terminally differentiated cells to preserve their lineage specificity. However, it is unclear whether mechanisms exist to actively protect cell identity in response to environmental cues that confer functional plasticity. Regulatory T (Treg) cells, specified by the transcription factor Foxp3, are indispensable for immune system homeostasis. Here, we report that conserved noncoding sequence 2 (CNS2), a CpG-rich Foxp3 intronic cis-element specifically demethylated in mature Tregs, helps maintain immune homeostasis and limit autoimmune disease development by protecting Treg identity in response to signals that shape mature Treg functions and drive their initial differentiation. In activated Tregs, CNS2 helps protect Foxp3 expression from destabilizing cytokine conditions by sensing TCR/NFAT activation, which facilitates the interaction between CNS2 and Foxp3 promoter. Thus, epigenetically marked cis-elements can protect cell identity by sensing key environmental cues central to both cell identity formation and functional plasticity without interfering with initial cell differentiation.

Purifying selection in deeply conserved human enhancers is more consistent than in coding sequences.

Comparison of polymorphism at synonymous and non-synonymous sites in protein-coding DNA can provide evidence for selective constraint. Non-coding DNA that forms part of the regulatory landscape presents more of a challenge since there is not such a clear-cut distinction between sites under stronger and weaker selective constraint. Here, we consider putative regulatory elements termed Conserved Non-coding Elements (CNEs) defined by their high level of sequence identity across all vertebrates. Some mutations in these regions have been implicated in developmental disorders; we analyse CNE polymorphism data to investigate whether such deleterious effects are widespread in humans. Single nucleotide variants from the HapMap and 1000 Genomes Projects were mapped across nearly 2000 CNEs. In the 1000 Genomes data we find a significant excess of rare derived alleles in CNEs relative to coding sequences; this pattern is absent in HapMap data, apparently obscured by ascertainment bias. The distribution of polymorphism within CNEs is not uniform; we could identify two categories of sites by exploiting deep vertebrate alignments: stretches that are non-variant, and those that have at least one substitution. The conserved category has fewer polymorphic sites and a greater excess of rare derived alleles, which can be explained by a large proportion of sites under strong purifying selection within humans – higher than that for non-synonymous sites in most protein coding regions, and comparable to that at the strongly conserved trans-dev genes. Conversely, the more evolutionarily labile CNE sites have an allele frequency distribution not significantly different from non-synonymous sites. Future studies should exploit genome-wide re-sequencing to obtain better coverage in selected non-coding regions, given the likelihood that mutations in evolutionarily conserved enhancer sequences are deleterious. Discovery pipelines should validate non-coding variants to aid in identifying causal and risk-enhancing variants in complex disorders, in contrast to the current focus on exome sequencing.

Classification of selectively constrained DNA elements using feature vectors and rule-based classifiers

Scarce work has been done in the analysis of the composition of conserved non-coding elements (CNEs) that are identified by comparisons of two or more genomes and are found to exist in all metazoan genomes.Here we present the analysis of CNEs with a methodology that takes into account word occurrence at various lengths scales in the form of feature vector representation and rule based classifiers. We implement our approach on both protein-coding exons and CNEs, originating from human, insect (Drosophila melanogaster) and worm (Caenorhabditis elegans) genomes, that are either identified in the present study or obtained from the literature.Alignment free feature vector representation of sequences combined with rule-based classification methods leads to successful classification of the different CNEs classes. Biologically meaningful results are derived by comparison with the genomic signatures approach, and classification rates for a variety of functional elements of the genomes along with surrogates are presented.

Inference of transcriptional networks in Arabidopsis through conserved noncoding sequence analysis.

Transcriptional regulation plays an important role in establishing gene expression profiles during development or in response to (a)biotic stimuli. Transcription factor binding sites (TFBSs) are the functional elements that determine transcriptional activity, and the identification of individual TFBS in genome sequences is a major goal to inferring regulatory networks. We have developed a phylogenetic footprinting approach for the identification of conserved noncoding sequences (CNSs) across 12 dicot plants. Whereas both alignment and non-alignment-based techniques were applied to identify functional motifs in a multispecies context, our method accounts for incomplete motif conservation as well as high sequence divergence between related species. We identified 69,361 footprints associated with 17,895 genes. Through the integration of known TFBS obtained from the literature and experimental studies, we used the CNSs to compile a gene regulatory network in Arabidopsis thaliana containing 40,758 interactions, of which two-thirds act through binding events located in DNase I hypersensitive sites. This network shows significant enrichment toward in vivo targets of known regulators, and its overall quality was confirmed using five different biological validation metrics. Finally, through the integration of detailed expression and function information, we demonstrate how static CNSs can be converted into condition-dependent regulatory networks, offering opportunities for regulatory gene annotation.

Identification of two regulatory elements controlling Fucosyltransferase 7 transcription in murine CD4+ T cells

Fucosyltransferase VII encoded by the gene Fut7 is essential in CD4+ T cells for the generation of E- and P-selectin ligands (E- and P-lig) which facilitate recruitment of lymphocytes into inflamed tissues and into the skin.This study aimed to identify regulatory elements controlling the inducible Fut7 expression in CD4+ T cells that occurs upon activation and differentiation of naive T cells into effector cells.Comparative analysis of the histone modification pattern in non-hematopoetic cells and CD4+ T cell subsets revealed a differential histone modification pattern within the Fut7 locus including a conserved non-coding sequence (CNS) identified by cross-species conservation comparison suggesting that regulatory elements are confined to this region.Cloning of the CNS located about 500bp upstream of the Fut7 locus, into a luciferase reporter vector elicited reporter activity after transfection of the αβ-WT T cell line, but not after transfection of primary murine CD4+ Th1 cells. As quantification of different Fut7 transcripts revealed a predominance of transcripts lacking the first exons in primary Th1 cells we searched for an alternative promoter. Cloning of an intragenic region spanning a 1kb region upstream of exon 4 into an enhancer-containing vector indeed elicited promoter activity. Interestingly, also the CNS enhanced activity of this intragenic minimal promoter in reporter assays in primary Th1 cells suggesting that both elements interact in primary CD4+ T cells to induce Fut7 transcription.

Sustained PI3K activation destabilized tTreg (BA15P.227)

Abstract Regulatory T cells (Treg) play a central role in immune homeostasis, whose dysfunction has been implicated in autoimmune disease pathogenesis. Two subsets Tregs have been defined in vivo: thymus derived tTregs and peripherally derived pTreg. The plasticity of Treg, especially the thymically derived tTreg remains considerable controversial. Since the conserved non-coding sequence 1(CNS1) of Foxp3 gene is only critical for the generation of induced pTreg cells, we generated a new BAC-transgenic mouse expressing a thy1.1 2A Cre fusion protein driven from the CNS1 deleted Foxp3 promoter (del CNS1 Foxp3-thy1.1-Cre). Characterization of these mice demonstrated the thy1.1 2A Cre fusion protein expressed exclusively in the Nrp1+Foxp3+ peripheral tTreg subset. Moreover, thy1.1 expression in the thymus was delayed compare with endogenous Foxp3. Stability of thy1.1+ Treg was tested by breeding the thy1.1 2A Cre Tg mouse line to a conditional YFP knock-in reporter mouse, termed ROSA26-stop-YFP. A subset range from 2% to 8% Cre mapped cells spontaneously lost Foxp3 expression in healthy animals, thus even the late developed tTreg cells have some extent of plasticity. Final, we found sustained PI3K activation by breeding with a conditional active PI3K Rosa26 knockin mouse, can significant destabilized late developed tTreg and generated pathogenic exFoxp3 cells in the homeostatic condition.

Runx Family Genes in a Cartilaginous Fish, the Elephant Shark (Callorhinchus milii)

The Runx family genes encode transcription factors that play key roles in hematopoiesis, skeletogenesis and neurogenesis and are often implicated in diseases. We describe here the cloning and characterization of Runx1, Runx2, Runx3 and Runxb genes in the elephant shark (Callorhinchus milii), a member of Chondrichthyes, the oldest living group of jawed vertebrates. Through the use of alternative promoters and/or alternative splicing, each of the elephant shark Runx genes expresses multiple isoforms similar to their orthologs in human and other bony vertebrates. The expression profiles of elephant shark Runx genes are similar to those of mammalian Runx genes. The syntenic blocks of genes at the elephant shark Runx gene loci are highly conserved in human, but represented by shorter conserved blocks in zebrafish indicating a higher degree of rearrangements in this teleost fish. Analysis of promoter regions revealed conservation of binding sites for transcription factors, including two tandem binding sites for Runx that are totally conserved in the distal promoter regions of elephant shark Runx1-3. Several conserved noncoding elements (CNEs), which are putative cis-regulatory elements, and miRNA binding sites were identified in the elephant shark and human Runx gene loci. Some of these CNEs and miRNA binding sites are absent in teleost fishes such as zebrafish and fugu. In summary, our analysis reveals that the genomic organization and expression profiles of Runx genes were already complex in the common ancestor of jawed vertebrates.

The Most Deeply Conserved Noncoding Sequences in Plants Serve Similar Functions to Those in Vertebrates Despite Large Differences in Evolutionary Rates

In vertebrates, conserved noncoding elements (CNEs) are functionally constrained sequences that can show striking conservation over >400 million years of evolutionary distance and frequently are located megabases away from target developmental genes. Conserved noncoding sequences (CNSs) in plants are much shorter, and it has been difficult to detect conservation among distantly related genomes. In this article, we show not only that CNS sequences can be detected throughout the eudicot clade of flowering plants, but also that a subset of 37 CNSs can be found in all flowering plants (diverging ∼170 million years ago). These CNSs are functionally similar to vertebrate CNEs, being highly associated with transcription factor and development genes and enriched in transcription factor binding sites. Some of the most highly conserved sequences occur in genes encoding RNA binding proteins, particularly the RNA splicing-associated SR genes. Differences in sequence conservation between plants and animals are likely to reflect differences in the biology of the organisms, with plants being much more able to tolerate genomic deletions and whole-genome duplication events due, in part, to their far greater fecundity compared with vertebrates.

The Histone Modification H3K27me3 Is Retained after Gene Duplication and Correlates with Conserved Noncoding Sequences in Arabidopsis

The histone modification H3K27me3 is involved in repression of transcription and plays a crucial role in developmental transitions in both animals and plants. It is deposited by PRC2 (Polycomb repressive complex 2), a conserved protein complex. In Arabidopsis thaliana, H3K27me3 is found at 15% of all genes. These tend to encode transcription factors and other regulators important for development. However, it is not known how PRC2 is recruited to target loci nor how this set of target genes arose during Arabidopsis evolution. To resolve the latter, we integrated A. thaliana gene families with five independent genome-wide H3K27me3 data sets. Gene families were either significantly enriched or depleted of H3K27me3, showing a strong impact of shared ancestry to H3K27me3 distribution. To quantify this, we performed ancestral state reconstruction of H3K27me3 on phylogenetic trees of gene families. The set of H3K27me3-marked genes changed less than expected by chance, suggesting that H3K27me3 was retained after gene duplication. This retention suggests that the PRC2-recruiting signal could be encoded in the DNA and also conserved among certain duplicated genes. Indeed, H3K27me3-marked genes were overrepresented among paralogs sharing conserved noncoding sequences (CNSs) that are enriched with transcription factor binding sites. The association of upstream CNSs with H3K27me3-marked genes represents the first genome-wide connection between H3K27me3 and potential regulatory elements in plants. Thus, we propose that CNSs likely function as part of the PRC2 recruitment in plants.

A Reporter Assay in Lamprey Embryos Reveals Both Functional Conservation and Elaboration of Vertebrate Enhancers

The sea lamprey is an important model organism for investigating the evolutionary origins of vertebrates. As more vertebrate genome sequences are obtained, evolutionary developmental biologists are becoming increasingly able to identify putative gene regulatory elements across the breadth of the vertebrate taxa. The identification of these regions makes it possible to address how changes at the genomic level have led to changes in developmental gene regulatory networks and ultimately to the evolution of morphological diversity. Comparative genomics approaches using sea lamprey have already predicted a number of such regulatory elements in the lamprey genome. Functional characterisation of these sequences and other similar elements requires efficient reporter assays in lamprey. In this report, we describe the development of a transient transgenesis method for lamprey embryos. Focusing on conserved non-coding elements (CNEs), we use this method to investigate their functional conservation across the vertebrate subphylum. We find instances of both functional conservation and lineage-specific functional evolution of CNEs across vertebrates, emphasising the utility of functionally testing homologous CNEs in their host species.

TGF-β-mediated Foxp3 gene expression is cooperatively regulated by Stat5, Creb, and AP-1 through CNS2.

Foxp3 plays an important role in the development and the function of regulatory T cells (Treg). Both the induction and maintenance of Foxp3 gene expression are controlled by several regulatory regions including two enhancers in the conserved noncoding sequences (CNS). The functions of Enhancer 1 in CNS1 are well established, whereas those of Enhancer 2 in CNS2 remain unclear. Although CNS2 contains enhancer activity, methylated CpG sequences in this region prevent Foxp3 gene expression in Foxp3(-) T cells. These sequences are, however, demethylated in Foxp3(+) Treg by mechanisms as yet unknown. To investigate the role of CNS2, we have determined the Enhancer 2 core sequence by luciferase reporter assays in the absence of methylation to exclude the inhibitory effect and shown that transcription factors AP-1, Stat5, and Creb cooperate in regulating Enhancer 2 activity. We have then determined the methylation sensitivity of each of the transcription factors. AP-1 was found to be methylation sensitive as has previously been described for Creb. However, Stat5 was active even when its binding site in CNS2 was methylated. Stat5 binding to Enhancer 2 occurred early and preceded that of AP-1 and Creb during Treg induction. In addition, Stat5 activation is itself dependent on TGF-β signaling through Smad3-mediated blockade of Socs3 expression. These findings suggest that Stat5 is a key regulator for opening up the CNS2 region during induced Treg induction, whereas AP-1 and Creb maintain Enhancer 2 activity.

A long non-coding RNA transcribed from conserved non-coding sequences contributes to the mouse prolyl oligopeptidase gene activation

Prolyl oligopeptidase (POP) is a multifunctional protease which is involved in many physiological events, but its gene regulatory mechanism is poorly understood. To identify novel regulatory elements of the POP gene, we compared the genomic sequences at the mouse and human POP loci and found six conserved non-coding sequences (CNSs) at adjacent intergenic regions. From these CNSs, four long non-coding RNAs (lncRNAs) were transcribed and the expression pattern of one (lncPrep+96kb) was correlated with that of POP. lncPrep+96kb was transcribed as two forms due to the different transcriptional start sites and was localized at the nucleus and cytoplasm, although more was present at the nucleus. When we knocked down lncPrep+96kb in the primary ovarian granulosa cell and a hepatic cell line, the POP expression was decreased in both cells. In contrast, overexpression of lncPrep+96kb increased the POP expression only in the granulosa cell. Because lncPrep+96kb was upregulated with the same timing as POP in the hormone-treated ovary, this lncRNA could play a role in the POP gene activation in the granulosa cell. Moreover, a downstream region of the human POP gene was also transcribed. We propose a novel mechanism for the POP gene activation.

Contributions of Protein-Coding and Regulatory Change to Adaptive Molecular Evolution in Murid Rodents

The contribution of regulatory versus protein change to adaptive evolution has long been controversial. In principle, the rate and strength of adaptation within functional genetic elements can be quantified on the basis of an excess of nucleotide substitutions between species compared to the neutral expectation or from effects of recent substitutions on nucleotide diversity at linked sites. Here, we infer the nature of selective forces acting in proteins, their UTRs and conserved noncoding elements (CNEs) using genome-wide patterns of diversity in wild house mice and divergence to related species. By applying an extension of the McDonald-Kreitman test, we infer that adaptive substitutions are widespread in protein-coding genes, UTRs and CNEs, and we estimate that there are at least four times as many adaptive substitutions in CNEs and UTRs as in proteins. We observe pronounced reductions in mean diversity around nonsynonymous sites (whether or not they have experienced a recent substitution). This can be explained by selection on multiple, linked CNEs and exons. We also observe substantial dips in mean diversity (after controlling for divergence) around protein-coding exons and CNEs, which can also be explained by the combined effects of many linked exons and CNEs. A model of background selection (BGS) can adequately explain the reduction in mean diversity observed around CNEs. However, BGS fails to explain the wide reductions in mean diversity surrounding exons (encompassing ∼100 Kb, on average), implying that there is a substantial role for adaptation within exons or closely linked sites. The wide dips in diversity around exons, which are hard to explain by BGS, suggest that the fitness effects of adaptive amino acid substitutions could be substantially larger than substitutions in CNEs. We conclude that although there appear to be many more adaptive noncoding changes, substitutions in proteins may dominate phenotypic evolution.

Parallel Evolution of Chordate Cis-Regulatory Code for Development

Urochordates are the closest relatives of vertebrates and at the larval stage, possess a characteristic bilateral chordate body plan. In vertebrates, the genes that orchestrate embryonic patterning are in part regulated by highly conserved non-coding elements (CNEs), yet these elements have not been identified in urochordate genomes. Consequently the evolution of the cis-regulatory code for urochordate development remains largely uncharacterised. Here, we use genome-wide comparisons between C. intestinalis and C. savignyi to identify putative urochordate cis-regulatory sequences. Ciona conserved non-coding elements (ciCNEs) are associated with largely the same key regulatory genes as vertebrate CNEs. Furthermore, some of the tested ciCNEs are able to activate reporter gene expression in both zebrafish and Ciona embryos, in a pattern that at least partially overlaps that of the gene they associate with, despite the absence of sequence identity. We also show that the ability of a ciCNE to up-regulate gene expression in vertebrate embryos can in some cases be localised to short sub-sequences, suggesting that functional cross-talk may be defined by small regions of ancestral regulatory logic, although functional sub-sequences may also be dispersed across the whole element. We conclude that the structure and organisation of cis-regulatory modules is very different between vertebrates and urochordates, reflecting their separate evolutionary histories. However, functional cross-talk still exists because the same repertoire of transcription factors has likely guided their parallel evolution, exploiting similar sets of binding sites but in different combinations.

Heterogeneous Tempo and Mode of Conserved Noncoding Sequence Evolution among Four Mammalian Orders

Conserved noncoding sequences (CNSs) of vertebrates are considered to be closely linked with protein-coding gene regulatory functions. We examined the abundance and genomic distribution of CNSs in four mammalian orders: primates, rodents, carnivores, and cetartiodactyls. We defined the two thresholds for CNS using conservation level of coding genes; using all the three coding positions and using only first and second codon positions. The abundance of CNSs varied among lineages, with primates and rodents having highest and lowest number of CNSs, respectively, whereas carnivores and cetartiodactyls had intermediate values. These CNSs cover 1.3–5.5% of the mammalian genomes and have signatures of selective constraints that are stronger in more ancestral than the recent ones. Evolution of new CNSs as well as retention of ancestral CNSs contribute to the differences in abundance. The genomic distribution of CNSs is dynamic with higher proportions of rodent and primate CNSs located in the introns compared with carnivores and cetartiodactyls. In fact, 19% of orthologous single-copy CNSs between human and dog are located in different genomic regions. If CNSs can be considered as candidates of gene expression regulatory sequences, heterogeneity of CNSs among the four mammalian orders may have played an important role in creating the order-specific phenotypes. Fewer CNSs in rodents suggest that rodent diversity is related to lower regulatory conservation. With CNSs shown to cluster around genes involved in nervous systems and the higher number of primate CNSs, our result suggests that CNSs may be involved in the higher complexity of the primate nervous system.

Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation

Intestinal microbes provide multicellular hosts with nutrients and confer resistance to infection. The delicate balance between pro- and anti-inflammatory mechanisms, essential for gut immune homeostasis, is affected by the composition of the commensal microbial community. Regulatory T (Treg) cells expressing transcription factor Foxp3 play a key role in limiting inflammatory responses in the intestine1. Although specific members of the commensal microbial community have been found to potentiate the generation of anti-inflammatory Treg or pro-inflammatory Th17 cells2-6, the molecular cues driving this process remain elusive. Considering the vital metabolic function afforded by commensal microorganisms, we hypothesized that their metabolic by-products are sensed by cells of the immune system and affect the balance between pro- and anti-inflammatory cells. We found that a short-chain fatty acid (SCFA), butyrate, produced by commensal microorganisms during starch fermentation, facilitated extrathymic generation of Treg cells. A boost in Treg cell numbers upon provision of butyrate was due to potentiation of extrathymic differentiation of Treg cells as the observed phenomenon was dependent upon intronic enhancer CNS1, essential for extrathymic, but dispensable for thymic Treg cell differentiation1, 7. In addition to butyrate, de novo Treg cell generation in the periphery was potentiated by propionate, another SCFA of microbial origin capable of HDAC inhibition, but not acetate, lacking this activity. Our results suggest that bacterial metabolites mediate communication between the commensal microbiota and the immune system, affecting the balance between pro- and anti-inflammatory mechanisms.