Conserved Noncoding Sequences Research Articles

Copy Number Variants in Two Northernmost Cattle Breeds Are Related to Their Adaptive Phenotypes.

Copy number variations (CNVs) are genomic structural variants with potential functional and evolutionary effects on phenotypes. In this study, we report the identification and characterization of CNVs from the whole-genome resequencing data of two northernmost cattle breeds from Russia: the Yakut and Kholmogory cattle and their phylogenetically most related breeds, Hanwoo and Holstein, respectively. Comparisons of the CNV regions (CNVRs) among the breeds led to the identification of breed-specific CNVRs shared by cold-adapted Kholmogory and Yakut cattle. An investigation of their overlap with genes, regulatory domains, conserved non-coding elements (CNEs), enhancers, and quantitative trait loci (QTLs) was performed to further explore breed-specific biology and adaptations. We found CNVRs enriched for gene ontology terms related to adaptation to environments in both the Kholmogory and Yakut breeds and related to thermoregulation specifically in Yakut cattle. Interestingly, the latter has also been supported when exploring the enrichment of breed-specific CNVRs in the regulatory domains and enhancers, CNEs, and QTLs implying the potential contribution of CNVR to the Yakut and Kholmogory cattle breeds’ adaptation to a harsh environment.

The Landscape of Liver Chromatin Accessibility and Conserved Non-coding Elements in Larimichthys crocea, Nibea albiflora, and Lateolabrax maculatus.

Large yellow croaker (Larimichthys crocea), yellow drum (Nibea albiflora), and Chinese seabass (Lateolabrax maculatus) are important economic marine fishes in China. The conserved non-coding elements (CNEs) in the liver tissues of the three kinds of fish are directly or indirectly involved in the regulation of gene expression and affect liver functions. However, the fishes' CNEs and even chromatin accessibility landscape have not been effectively investigated. Hence, this study established the landscapes of the fishes' genome-wide chromatin accessibility and CNEs by detecting regions of the open chromatin in their livers using an assay for transposase-accessible chromatin by high-throughput sequencing (ATAC-seq) and comparative genomics approach. The results showed that Smad1, Sp1, and Foxl1 transcription factor binding motifs were considerably enrichedin thechromatin accessibility landscape in theliver of the three species, and the three transcription factors (TFs) had a wide range of common targets. The hypothetical gene set was targeted by one, two, or all three TFs, which was much higher than would be expected for an accidental outcome. The gene sets near the CNEs were mainly enriched through processes such as a macromolecule metabolic process and ribonucleoprotein complex biogenesis. The active CNEs were found in the promoter regions of genes such as ap1g1, hax1, and ndufs2. And 5 CNEs were predicted to be highly conserved active enhancers. These results demonstrated that Smad1, Sp1, and Foxl1 might be related to the liver function in the three fishes. In addition, we found a series of ATAC-seq-labeled CNEs located in the gene promoter regions, and highly conserved H3k27ac + -labeled CNEs located in the liver function genes. The highly conserved nature of these regulatory elements suggests that they play important roles in the liver in fish. This study minedthelandscape of chromatin accessibility and CNEs of three important economic fishes to fill the knowledge gaps in this field. Moreover, the work provides useful data for the industrial application and theoretical research of these three fish species.

Role of CNSs Conserved Distal Cis-Regulatory Elements in CD4 + T Cell Development and Differentiation.

Naïve CD4+ T cells differentiate into diverse subsets of effector cells and perform various homeostatic and immune functions. The differentiation and maintenance of these different subsets are controlled through the upregulation and silencing of master genes. Mechanistic studies of the regulation of these master genes identified conserved and distal intronic regulatory elements, which are accessible subsets of conserved non-coding sequences (CNSs), acting as cis-regulatory elements in a lineage-specific manner that controls the function of CD4+ T cells. Abnormal CNS activity is associated with incorrect expression of master genes and development of autoimmune diseases or immune suppression. Here, we describe the function of several conserved, distal cis-regulatory elements at the Foxp3, Rorc, Il-4, Il-10 and Il-17 gene locus were shown to play important roles in CD4+ T cells differentiation. Together, this review briefly outlines currently known CNSs, with a focus on their regulations and functions in complexes modulating the differentiation and maintenance of various CD4+ T cells subsets, in health and disease contexts, as well as during the conversion of T regulatory cells to T helper 17 cells. This article will provide a comprehensive view of CNSs conserved distal cis-regulatory elements at a few loci that control aspects of CD4+ T cells function.

Systematic annotation of conservation states provides insights into regulatory regions in rice

Plant genomes contain a large fraction of noncoding sequences. The discovery and annotation of conserved noncoding sequences (CNSs) in plants is an ongoing challenge. Here we report the application of comparative genomics to systematically identify CNSs in 50 well-annotated Gramineae genomes using rice (Oryza sativa) as the reference. We conduct multiple-way whole-genome alignments to the rice genome. The rice genome is annotated as 20 conservation states (CSs) at single-nucleotide resolution using a multivariate hidden Markov model (ConsHMM) based on the multiple-genome alignments. Different states show distinct enrichments for various genomic features, and the conservation scores of CSs are highly correlated with the level of associated chromatin accessibility. We find that at least 33.5% of the rice genome is highly under selection, with more than 70% of the sequence lying outside of coding regions. A catalog of 855,366 regulatory CNSs is generated, and they significantly overlapped with putative active regulatory elements such as promoters, enhancers, and transcription factor binding sites. Collectively, our study provides a resource for elucidating functional noncoding regions of the rice genome and an evolutionary aspect of regulatory sequences in higher plants.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade.

Summary Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen‐fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade.To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen‐fixing clade (NFC).We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1.CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

Copy number variations residing outside the SHOX enhancer region are involved in Short Stature and Léri-Weill dyschondrosteosis.

Background SHOX enhancer CNVs, affecting one or more of the seven recognized evolutionary conserved non‐coding elements (CNEs) represent one of the most frequent cause of SHOX‐haploinsufficiency. During the diagnostic workflow deletions/duplications have been identified downstream SHOX not including any of the these CNEs.MethodsFine tiling aCGH and breakpoint PCR were used to characterize the critical interval and to search for novel alterations in a cohort of selected patients.ResultsScreening of 252 controls provided evidence that duplications in this area represent likely benign variants whereas none of the deletions were detected. These findings suggested that other alterations relevant for SHOX‐haploinsufficiency might be missed by the standard diagnostic methods. To identify such undisclosed elements, the aCGH was used to reanalyze 52 unresolved cases with clinical features strongly suggestive of SHOX‐haploinsufficiency. This analysis followed by the screening of 210 patients detected two partially overlapping small deletions of ~12 and ~8 kb in four unrelated individuals, approximately 15 kb downstream SHOX, that were absent in 720 normal stature individuals.ConclusionOur results strengthen the hypothesis that alterations of yet unidentified cis‐regulatory elements residing outside those investigated through conventional methods, might explain the phenotype in ISS/LWD patients thus enlarging the spectrum of variants contributing to SHOX‐haploinsufficiency.

The role of IL-4 derived from follicular helper T (TFH) cells and type 2 helper T (TH2) cells.

IL-4 is known to be the quintessential regulatory cytokine, playing a role in a vast number of immune and non-immune functions. This cytokine is commonly secreted by type 2 helper T (TH2) cells and follicular helper T (TFH) cells after antigenic sensitization. TH2 cells have been classically thought to be the major contributor to B-cell help as a source of IL-4 responsible for class-switch recombination to IgG1 in mice (IgG4 in humans) and to IgE in mice and humans. Recent in vivo observations have shown that IgE and IgG1 antibody responses are mainly controlled by IL-4-secreting TFH cells but not by classical TH2 cells. IL-4 is distinctively regulated in these two T-cell subsets by the GATA-3-mediated HS2 enhancer in TH2 cells and the Notch-mediated conserved non-coding sequence 2 (CNS-2) enhancer in TFH cells. Moreover, the IL-4 derived from TFH cells has an essential role in germinal center (GC) formation in the secondary lymphoid organs during humoral immune responses.

Dynamics and epigenetic signature of regulatory T-cells following antiretroviral therapy initiation in acute HIV infection.

BackgroundHIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied.MethodsTreg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology.FindingsTotal Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-β1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-β7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-β1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-β1)+ Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-β1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different.InterpretationEarly ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression.FundingThis study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Development of a Treg Lineage Specific Lentiviral Vector for the Treatment of IPEX Syndrome

▪Autologous hematopoietic stem cell (HSC) transplant with gene therapy has recently demonstrated remarkable clinical success in the correction of genetic blood cell disorders. One excellent candidate disease for HSC gene therapy is Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome. IPEX is a devastating autoimmune disease caused by mutations in FOXP3, a transcription factor required for the proper development and function of regulatory T (Treg) cells. Tregs are critical for providing dominant suppression of autoreactive conventional T (Tconv) cells, and thus their absence in IPEX patients causes severe and often fatal multi-organ autoimmunity. Allogeneic HSC transplant has been shown to be curative for IPEX patients, but is limited due to a lack of suitable donors and immunologic transplant complications. In order to explore an autologous HSC gene therapy strategy for IPEX, we sought to design an endogenously regulated lentiviral vector (LV) which confers targeted and developmentally appropriate FoxP3 expression. Here, we utilized three intronic regulatory regions of the FoxP3 gene which have verified enhancer/regulatory function: conserved noncoding sequence (CNS) 1, CNS2, and CNS3. These sequences were added upstream from the FoxP3 promoter and cloned into the CCL vector backbone to generate two LV expressing either mStrawberry (FoxP3 reporter vector), or a human FoxP3 cDNA and mStrawberry (FoxP3 cDNA vector). A primary screen of the FoxP3 reporter vector in human cell lines from different hematopoietic lineages (K562 [erythromyeloid, FoxP3-], Jurkat [Tconv, FoxP3-] and MT-2 [Treg-like, FoxP3+]) revealed mStrawberry expression restricted to the FoxP3+ cell line with low inherent “leakiness” of expression in FoxP3- cell types. In order to evaluate in vivo lineage specificity of the FoxP3 reporter vector, we utilized both murine congenic transplants and humanized NSG xenografts to quantify LV expression in transduced HSPC and their progeny throughout hematopoietic development. In both models, we observed high levels of mStrawberry expression in the Treg lineage with low expression in CD4 Tconv cells and CD8 T cells, and negligible expression in HSPC, B cells, myeloid cells, suggesting that the regulatory elements included in this vector confer developmentally appropriate FoxP3 expression. We next evaluated the capacity of this vector to restore Treg development when introduced into FoxP3-deficient HSC from the scurfy mouse. Scurfy HSC were transduced with the FoxP3 cDNA vector and transplanted into WT CD45.1 congenic recipients to generate corrected scurfy Tregs (cSf-Tregs). Thymic cSf-Tregs phenotypically resembled WT-Treg showing equally high expression of the Treg surface markers CD25, GITR, and CTLA-4. Furthermore, FACS-sorted cSf-Tregs were equivalent to WT-Tregs in their ability to suppress Tconv cell proliferation in vitro. We next evaluated the in vivo functionality of cSf-Tregs by assessing their ability to reverse clinical disease signs in neonatal scurfy mice. Donor CD45.2+CD4+ cells (containing either WT-Tregs, cSf-Tregs, or uncorrected Sf T cells) were purified from the spleens of WT CD45.1 transplant recipients and administered to scurfy neonates by i.p. injection. Treated scurfy mice were evaluated for disease progression at 21 days post-injection. In comparison to WT littermate controls, untreated scurfy mice showed typical signs of disease progression including scaly skin, malformed ears, splenomegaly, and a high percentage of activated (CD62L-CD44+) CD4 T cells. Administration of WT-Tregs or cSf-Tregs resulted in complete correction of all measures of the scurfy phenotype, such that these mice were indistinguishable from WT littermate controls. In contrast, scurfy mice receiving uncorrected Sf T cells showed no signs of disease amelioration, confirming that disease correction was not an artifact of the transplant/CD4 isolation procedure. These results demonstrate that correction of scurfy HSC with a lineage-specific FoxP3 cDNA LV produces CD4+ Tregs capable of controlling autoimmune T cell responses in vivo. These findings pave the way for the treatment of IPEX patients by autologous HSC transplant and may further provide valuable insights into new treatments for patients with autoimmune disorders of different origin. DisclosuresKohn:Orchard Therapeutics Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Research Funding; Biogen IDEC: Research Funding.

Conserved noncoding sequences provide insights into regulatory sequence and loss of gene expression in maize.

Thousands of species will be sequenced in the next few years; however, understanding how their genomes work, without an unlimited budget, requires both molecular and novel evolutionary approaches. We developed a sensitive sequence alignment pipeline to identify conserved noncoding sequences (CNSs) in the Andropogoneae tribe (multiple crop species descended from a common ancestor ∼18 million years ago). The Andropogoneae share similar physiology while being tremendously genomically diverse, harboring a broad range of ploidy levels, structural variation, and transposons. These contribute to the potential of Andropogoneae as a powerful system for studying CNSs and are factors we leverage to understand the function of maize CNSs. We found that 86% of CNSs were comprised of annotated features, including introns, UTRs, putative cis-regulatory elements, chromatin loop anchors, noncoding RNA (ncRNA) genes, and several transposable element superfamilies. CNSs were enriched in active regions of DNA replication in the early S phase of the mitotic cell cycle and showed different DNA methylation ratios compared to the genome-wide background. More than half of putative cis-regulatory sequences (identified via other methods) overlapped with CNSs detected in this study. Variants in CNSs were associated with gene expression levels, and CNS absence contributed to loss of gene expression. Furthermore, the evolution of CNSs was associated with the functional diversification of duplicated genes in the context of maize subgenomes. Our results provide a quantitative understanding of the molecular processes governing the evolution of CNSs in maize.

Distinct Foxp3 enhancer elements coordinate development, maintenance, and function of regulatory T cells

The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.

Cutting Edge: CRISPR-Based Transcriptional Regulators Reveal Transcription-Dependent Establishment of Epigenetic Memory of Foxp3 in Regulatory T Cells.

Transcription factor Foxp3 specifies and maintains regulatory T cell (Treg) identity. During Treg differentiation, a CpG-rich Foxp3 intronic enhancer, conserved noncoding sequence 2 (CNS2), is activated via DNA demethylation to establish epigenetic memory of Foxp3 expression to protect Treg identity. However, it is unclear how this epigenetic memory of Foxp3 expression is established, as CNS2 is thought to be demethylated independently of Foxp3 expression. In this article, we uncover an unexpected causal relationship between Foxp3-transcriptional activation and CNS2 demethylation in mice. CRISPR/dCas9-mediated Foxp3-transcriptional activation elicits CNS2 demethylation. Sustaining Foxp3-transcriptional activation in induced Tregs also promotes CNS2 demethylation, enhancing Treg lineage stability and suppressive function. Importantly, CRISPR-mediated silencing of Foxp3 transcription, but not protein expression, abolishes CNS2 demethylation. The novel finding that Foxp3-transcriptional activation promotes CNS2 demethylation may facilitate the development of Treg-based therapies and represent a general mechanism for the establishment of epigenetic memory of immune gene expression.

DbCNS: A New Database for Conserved Noncoding Sequences.

We developed dbCNS (http://yamasati.nig.ac.jp/dbcns), a new database for conserved noncoding sequences (CNSs). CNSs exist in many eukaryotes and are assumed to be involved in protein expression control. Version 1 of dbCNS, introduced here, includes a powerful and precise CNS identification pipeline for multiple vertebrate genomes. Mutations in CNSs may induce morphological changes and cause genetic diseases. For this reason, many vertebrate CNSs have been identified, with special reference to primate genomes. We integrated ∼6.9 million CNSs from many vertebrate genomes into dbCNS, which allows users to extract CNSs near genes of interest using keyword searches. In addition to CNSs, dbCNS contains published genome sequences of 161 species. With purposeful taxonomic sampling of genomes, users can employ CNSs as queries to reconstruct CNS alignments and phylogenetic trees, to evaluate CNS modifications, acquisitions, and losses, and to roughly identify species with CNSs having accelerated substitution rates. dbCNS also produces links to dbSNP for searching pathogenic single-nucleotide polymorphisms in human CNSs. Thus, dbCNS connects morphological changes with genetic diseases. A test analysis using 38 gnathostome genomes was accomplished within 30 s. dbCNS results can evaluate CNSs identified by other stand-alone programs using genome-scale data.

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

Immunomodulatory Effects of Calcitriol through DNA Methylation Alteration of FOXP3 in the CD4+ T Cells of Mice.

Vitamin D plays a variety of physiological functions, such as regulating mineral homeostasis. More recently, it has emerged as an immunomodulator player, affecting several types of immune cells, such as regulatory T (Treg) cells. It has been reported that vitamin D exerts some mediatory effects through an epigenetic mechanism. In this study, the impacts of calcitriol, the active form of vitamin D, on the methylation of the conserved non-coding sequence 2 (CNS2) region of the forkhead box P3 (Foxp3) gene promoter, were evaluated. Fourteen C57BL/6 mice were recruited in this study and divided into two intervention and control groups. The CD4+ T cells were isolated from mice splenocytes. The expression of Foxp3, IL-10, and transforming growth factor-beta (TGF-β1) genes were relatively quantified by real-time PCR technique, and the DNA methylation percentage of every CpG site in the CNS2 region was measured individually by bisulfite-sequencing PCR. Vitamin D Intervention significantly (p<0.05) could increase the expression of Foxp3, IL-10, and TGF-β1 gene in the CD4+ T cells of mice comparing with the control group. Meanwhile, methylation of the CNS2 region of Foxp3 promoter was significantly decreased in three of ten CpG sites in the vitamin D group compared to the control group. The results of this study showed that vitamin D can engage the methylation process to induce Foxp3 gene expression and probably Treg cytokines profile. Further researches are needed to discover the precise epigenetic mechanisms by which vitamin D modulates the immune system.

Expansion of the circadian transcriptome in Brassica rapa and genome-wide diversification of paralog expression patterns.

An important challenge of crop improvement strategies is assigning function to paralogs in polyploid crops. Here we describe the circadian transcriptome in the polyploid crop Brassica rapa. Strikingly, almost three-quarters of the expressed genes exhibited circadian rhythmicity. Genetic redundancy resulting from whole genome duplication is thought to facilitate evolutionary change through sub- and neo-functionalization among paralogous gene pairs. We observed genome-wide expansion of the circadian expression phase among retained paralogous pairs. Using gene regulatory network models, we compared transcription factor targets between B. rapa and Arabidopsis circadian networks to reveal evidence for divergence between B. rapa paralogs that may be driven in part by variation in conserved non-coding sequences (CNS). Additionally, differential drought response among retained paralogous pairs suggests further functional diversification. These findings support the rapid expansion and divergence of the transcriptional network in a polyploid crop and offer a new approach for assessing paralog activity at the transcript level.

The Conserved Non-coding Sequences CNS6 and CNS9 Control Cytokine-Induced Rorc Transcription during T Helper 17 Cell Differentiation

RORγt is the lineage-specific transcription factor for T helper 17 (Th17) cells whose upregulation in developing Th17 cells is critically regulated by interleukin-6 (IL-6) and TGF-β, the molecular mechanisms of which remain largely unknown. Here we identified conserved non-coding sequences (CNSs) 6 and 9 at the Rorc gene, essential for its expression during Th17 cell differentiation but not required for RORγt expression in innate lymphocytes and γδ Tcells. Mechanistically, the IL-6-signal transducer and activator of transcription 3 (STAT3) axis appeared to be largely dependent on CNS9 and only partially on CNS6 in controlling RORγt expression and epigenetic activation of the Rorc locus. TGF-β alone was sufficient to induce RORγt expression in a CNS6- but not CNS9-dependent manner through CNS6 binding by SMAD proteins. Our study reveals an important synergistic mechanism downstream of IL-6 and TGF-β in regulation of RORγt expression and Th17 cell commitment via distinct cis-regulatory elements.

Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia

BackgroundMicrotia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1 transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number variations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes.MethodsSingle Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2 were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function. qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia.ResultsLinkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in coding regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions in 61 patients with other type of microtia.ConclusionDuplications involving long range HMX1 enhancers are associated with human isolated bilateral concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear malformations, as in other species. This study also provides an additional example of functional conserved non-coding elements (CNEs) in humans.

Eomesodermin promotes interaction of RelA and NFATc2 with the Ifng promoter and multiple conserved noncoding sequences across the Ifng locus in mouse lymphoma BW5147 cells

The T-box transcription factor Eomesodermin (Eomes) regulates the lineage-dependent expression of interferon γ (IFN-γ). We previously showed that Eomes promotes IFN-γ production and interacts with multiple conserved noncoding sequences (CNS) across the Ifng locus in mouse lymphoma BW5147 cells. In the present study, we investigated the transcriptional regulation of IFN-γ by the nuclear factor κB (NF-κB) subunit RelA and nuclear factor of activated T cells c2 (NFATc2, also known as NFAT1) in Eomes-transfected BW5147 cells. Eomes promoted the interaction of RelA and NFATc2 with the Ifng promoter and five CNS, including CNS-22 and CNS+30 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). The dual NF-κB and STAT3 inhibitor TPCA-1 moderately reduced the PMA- and IM-induced IFN-γ transcription in Eomes-transfected BW5147 cells. TPCA-1 interfered with RelA binding to the Ifng promoter, CNS-22 and CNS+30. Moreover, TPCA-1 reduced the interaction of Eomes or NFATc2 with the Ifng promoter and CNS+30. The present results indicate that Eomes promotes the interaction of RelA and NFATc2 with the Ifng promoter and multiple CNS across the Ifng locus in BW5147 cells.

Draft genomes of two outcrossing wild rice, Oryza rufipogon and O. longistaminata, reveal genomic features associated with mating-system evolution.

Oryza rufipogon and O. longistaminata are important wild relatives of cultivated rice, harboring a promising source of novel genes for rice breeding programs. Here, we present de novo assembled draft genomes and annotation of O. rufipogon and O. longistaminata. Our analysis reveals a considerable number of lineage‐specific gene families associated with the self‐incompatibility (SI) and formation of reproductive separation. We show how lineage‐specific expansion or contraction of gene families with functional enrichment of the recognition of pollen, thus enlightening their reproductive diversification. We documented a large number of lineage‐specific gene families enriched in salt stress, antifungal response, and disease resistance. Our comparative analysis further shows a genome‐wide expansion of genes encoding NBS‐LRR proteins in these two outcrossing wild species in contrast to six other selfing rice species. Conserved noncoding sequences (CNSs) in the two wild rice genomes rapidly evolve relative to selfing rice species, resulting in the reduction of genomic variation owing to shifts of mating systems. We find that numerous genes related to these rapidly evolving CNSs are enriched in reproductive structure development, flower development, and postembryonic development, which may associate with SI in O. rufipogon and O. longistaminata.