The NF-κB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel(-/-)), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel(-/-) mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.

Plasticity of DNA methylation in mouse T cell activation and differentiation.

BACKGROUND: Circulating CD4+ T helper cells are activated through interactions with antigen presenting cells and undergo differentiation into specific T helper cell subsets depending on the type of antigen encountered. In addition, the relative composition of the circulating CD4+ T cell population changes as animals mature with an increased percentage of the population being memory/effector type cells. RESULTS: Here, we report on the highly plastic nature of DNA methylation at the genome-wide level as T cells undergo activation, differentiation and aging. Of particular note were the findings that DNA demethylation occurred rapidly following T cell activation and that all differentiated T cell populations displayed lower levels of global methylation than the non-differentiated population. In addition, T cells from older mice had a reduced level of DNA methylation, most likely explained by the increase in the memory/effector cell fraction. Although significant genome-wide changes were observed, changes in DNA methylation at individual genes were restricted to specific cell types. Changes in the expression of enzymes involved in DNA methylation and demethylation reflect in most cases the changes observed in the genome-wide DNA methylation status. CONCLUSION: We have demonstrated that DNA methylation is dynamic and flexible in CD4+ T cells and changes rapidly both in a genome-wide and in a targeted manner during T cell activation, differentiation. These changes are accompanied by parallel changes in the enzymatic complexes that have been implicated in DNA methylation and demethylation implying that the balance between these opposing activities may play a role in the maintaining the methylation profile of a given cell type but also allow flexibility in a cell population that needs to respond rapidly to environmental signals.

Regulation of the IL-21 gene by the NF-κB transcription factor c-Rel.

IL-21 is a member of the common gamma-chain-dependent cytokine family and is a key modulator of lymphocyte development, proliferation, and differentiation. IL-21 is highly expressed in activated CD4(+) T cells and plays a critical role in the expansion and differentiation of the Th cell subsets, Th17 and follicular helper T (T(FH)) cells. Because of its potent activity in both myeloid and lymphoid cell immune responses, it has been implicated in a number of autoimmune diseases and has also been used as a therapeutic agent in the treatment of some cancers. In this study, we demonstrate that c-Rel, a member of the NF-kappaB family of transcription factors, is required for IL-21 gene expression in T lymphocytes. IL-21 mRNA and protein levels are reduced in the CD4(+) cells of rel(-/-) mice when compared with rel(+/+) mice in both in vitro and in vivo models. A c-Rel binding site identified in the proximal promoter of il21 is confirmed to bind c-Rel in vitro and in vivo and to regulate expression from the il21 promoter in T cells. Downstream of IL-21 expression, Th17, T(FH), and germinal center B cell development are also impaired in rel(-/-) mice. The administration of IL-21 protein rescued the development of T(FH) cells but not germinal center B cells. Taken together, c-Rel plays an important role in the expression of IL-21 in T cells and subsequently in IL-21-dependent T(FH) cell development.

Genome-wide identification of human FOXP3 target genes in natural regulatory T cells.

The transcription factor FOXP3 is essential for the formation and function of regulatory T cells (Tregs), and Tregs are essential for maintaining immune homeostasis and tolerance. This is demonstrated by a lethal autoimmune defect in mice lacking Foxp3 and in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome patients. However, little is known about the molecular basis of human FOXP3 function or the relationship between direct and indirect targets of FOXP3 in human Tregs. To investigate this, we have performed a comprehensive genome-wide analysis for human FOXP3 target genes from cord blood Tregs using chromatin immunoprecipitation array profiling and expression profiling. We have identified 5579 human FOXP3 target genes and derived a core Treg gene signature conserved across species using mouse chromatin immunoprecipitation data sets. A total of 739 of the 5579 FOXP3 target genes were differentially regulated in Tregs compared with Th cells, thus allowing the identification of a number of pathways and biological functions overrepresented in Tregs. We have identified gene families including cell surface molecules and microRNAs that are differentially expressed in FOXP3(+) Tregs. In particular, we have identified a novel role for peptidase inhibitor 16, which is expressed on the cell surface of >80% of resting human CD25(+)FOXP3(+) Tregs, suggesting that in conjunction with CD25 peptidase inhibitor 16 may be a surrogate surface marker for Tregs with potential clinical application.

Changes in chromatin accessibility across the GM-CSF promoter upon T cell activation are dependent on nuclear factor kappaB proteins.

Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.

The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells.

Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor kappaB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter.

Interindividual variation in epigenomic phenomena in humans.

Our knowledge of regulatory mechanisms of gene expression and other chromosomal processes related to DNA methylation and chromatin state is continuing to grow at a rapid pace. Understanding how these epigenomic phenomena vary between individuals will have an impact on understanding their broader role in determining variation in gene expression and biochemical, physiological, and behavioural phenotypes. In this review we survey recent progress in this area, focusing on data available from humans. We highlight the role of obligatory (sequence-dependent) epigenomic variation as an important mechanism for generating interindividual variation that could impact our understanding of the mechanistic basis of complex trait architecture.

Histone dynamics on the interleukin-2 gene in response to T-cell activation.

Several models have been proposed for the mechanism of chromatin remodelling across the promoters of inducible genes in mammalian cells. The most commonly held model is one of cooccupation where histone proteins are modified by acetylation or phosphorylation and nucleosomes are remodelled, allowing the assembly of transcription factor complexes. Using chromatin immunoprecipitation, we observed an apparent decrease of histone acetylation and phosphorylation signals at the proximal promoter region of the inducible interleukin-2 and granulocyte-macrophage colony-stimulating factor genes in response to T-cell activation. We showed that this apparent decrease was due to a loss of histone H3 and H4 proteins corresponding to a decrease in nucleosome occupation of the promoter. This histone loss is reversible; it is dependent on the continual presence of appropriate activating signals and transcription factors and is not dependent on the acetylation status of the histone proteins. These data show for the first time that histone proteins are lost from a mammalian promoter upon activation of transcription and support a model of activation-dependent disassembly and reassembly of nucleosomes.

GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements.

Granulocyte-macrophage colony stimulating factor (GM-CSF) plays a key role in myeloid cell function and is rapidly and transiently expressed in T cells in response to immune or inflammatory stimuli. Induction of GM-CSF gene expression is accompanied by changes in chromatin structure across the proximal promoter region of the gene. We show that the promoter remodelling and subsequent gene transcription occurs with distinct signal and transcription factor requirements. Activation of the protein kinase C (PKC) signalling pathway is sufficient to induce changes in chromatin structure across the promoter, but both the PKC and calcium signalling pathways are required for efficient gene transcription. Although NFAT transcription factors contribute to GM-CSF gene transcription, they are not required for promoter remodelling. However, the presence of the nuclear factor-kappaB transcription factor, c-Rel, in the nucleus is strongly correlated with and required for the events of chromatin remodelling.

Changes in the expression of miR-381 and miR-495 are inversely associated with the expression of the MDR1 gene and development of multi-drug resistance.

Multidrug resistance (MDR) frequently develops in cancer patients exposed to chemotherapeutic agents and is usually brought about by over-expression of P-glycoprotein (P-gp) which acts as a drug efflux pump to reduce the intracellular concentration of the drug(s). Thus, inhibiting P-gp expression might assist in overcoming MDR in cancer chemotherapy. MiRNAome profiling using next-generation sequencing identified differentially expressed microRNAs (miRs) between parental K562 cells and MDR K562 cells (K562/ADM) induced by adriamycin treatment. Two miRs, miR-381 and miR-495, that were strongly down-regulated in K562/ADM cells, are validated to target the 3'-UTR of the MDR1 gene. These miRs are located within a miR cluster located at chromosome region 14q32.31, and all miRs in this cluster appear to be down-regulated in K562/ADM cells. Functional analysis indicated that restoring expression of miR-381 or miR-495 in K562/ADM cells was correlated with reduced expression of the MDR1 gene and its protein product, P-gp, and increased drug uptake by the cells. Thus, we have demonstrated that changing the levels of certain miR species modulates the MDR phenotype in leukemia cells, and propose further exploration of the use of miR-based therapies to overcome MDR.

An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a form of cellular plasticity that is critical for embryonic development and tumor metastasis. A double-negative feedback loop involving the miR-200 family and ZEB (zinc finger E-box-binding homeobox) transcription factors has been postulated to control the balance between epithelial and mesenchymal states. Here we demonstrate using the epithelial Madin Darby canine kidney cell line model that, although manipulation of the ZEB/miR-200 balance is able to repeatedly switch cells between epithelial and mesenchymal states, the induction and maintenance of a stable mesenchymal phenotype requires the establishment of autocrine transforming growth factor-ß (TGF-ß) signaling to drive sustained ZEB expression. Furthermore, we show that prolonged autocrine TGF-ß signaling induced reversible DNA methylation of the miR-200 loci with corresponding changes in miR-200 levels. Collectively, these findings demonstrate the existence of an autocrine TGF-ß/ZEB/miR-200 signaling network that regulates plasticity between epithelial and mesenchymal states. We find a strong correlation between ZEBs and TGF-ß and negative correlations between miR-200 and TGF-ß and between miR-200 and ZEBs, in invasive ductal carcinomas, consistent with an autocrine TGF-ß/ZEB/miR-200 signaling network being active in breast cancers.

Epigenetic control of inducible gene expression in the immune system.

It has been well documented that active genes, and their promoters and enhancers have a different chromatin or epigenomic environment compared with unexpressed genes. In addition, the epigenome may influence not only which genes are expressed, but also which genes can be induced in response to activation or differentiation signals. Immune cells respond to activation signals by rapidly inducing the expression of specific gene sets, and therefore this is a good system in which to examine the role of the epigenome in gene activation and cell differentiation. Several studies have now found that many immediate-early inducible genes exist in a similar epigenomic environment to active genes even in the unstimulated state. Some studies suggest that subsets of these genes may even have RNA polymerase II at their promoters and induction may be controlled downstream of its recruitment. Other inducible genes, however, undergo changes to histone modifications, levels or variant composition upon activation. In this article, we discuss how the epigenome of immune cells regulates inducible gene expression and discuss the differences between the immediate responses to activation signals and the longer term changes observed during differentiation.

The impact of CpG island on defining transcriptional activation of the mouse L1 retrotransposable elements.

BACKGROUND: L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active T(F) family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome. CONCLUSIONS: A comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements.

Development of CD4+CD25+FoxP3+ regulatory T cells from cord blood hematopoietic progenitor cells.

Adult stem cells are capable of generating all of the cells of the hematopoietic system, and this process is orchestrated in part by the interactions between these cells and the stroma. T cell progenitors emerge from the stem cell compartment and migrate to the thymus, where their terminal differentiation and maturation occur, and it is during this phase that selection shapes the immune repertoire. Notch ligands, including Delta-like 1 (DL1), play a critical role in this lymphoid differentiation. To mimic this in vitro, stroma-expressing DL1 have been used to generate CD4(+)CD8(+) double-positive and single-positive T cells from hematopoietic stem/progenitor cells. This system provides a robust tool to investigate thymopoiesis; however, its capacity to generate regulatory T cells (Tregs) has yet to be reported. Natural Tregs (nTregs) develop in the thymus and help maintain immune homeostasis and have potential clinical use as a cell therapy for modulation of autoimmune disease or for transplant tolerization. Here, we describe for the first time the development of a population of CD4(+)CD25(+) CD127(lo)FoxP3(+) cells that emerge in coculture of cord blood (CB) CD34(+) progenitors on OP9-DL1 stroma. These hematopoietic progenitor-derived CD4(+)CD25(+) Tregs have comparable suppressor function with CB nTregs in vitro. The addition of IL-2 to the coculture enhanced the expansion and survival of this population significantly. This manipulable culture system, therefore, generates functional Tregs and provides a system to elucidate the mechanism of Treg development.

c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells.

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-kappaB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel(-/-) mice, thymic T reg cell numbers are markedly reduced as a result of a T cell-intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-beta conversion of peripheral CD4(+)CD25(-) T cells into CD4(+)Foxp3(+) cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel(-/-) mice, the residual peripheral c-rel(-/-) T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

A double-negative feedback loop between ZEB1-SIP1 and the microRNA-200 family regulates epithelial-mesenchymal transition.

Epithelial to mesenchymal transition occurs during embryologic development to allow tissue remodeling and is proposed to be a key step in the metastasis of epithelial-derived tumors. The miR-200 family of microRNAs plays a major role in specifying the epithelial phenotype by preventing expression of the transcription repressors, ZEB1/deltaEF1 and SIP1/ZEB2. We show here that miR-200a, miR-200b, and the related miR-429 are all encoded on a 7.5-kb polycistronic primary miRNA (pri-miR) transcript. We show that the promoter for the pri-miR is located within a 300-bp segment located 4 kb upstream of miR-200b. This promoter region is sufficient to confer expression in epithelial cells and is repressed in mesenchymal cells by ZEB1 and SIP1 through their binding to a conserved pair of ZEB-type E-box elements located proximal to the transcription start site. These findings establish a double-negative feedback loop controlling ZEB1-SIP1 and miR-200 family expression that regulates cellular phenotype and has direct relevance to the role of these factors in tumor progression.

Genome-wide analysis of gene expression in T cells to identify targets of the NF-kappa B transcription factor c-Rel.

It is well established that the NF-kappaB family of transcription factors serves a major role in controlling gene expression in response to T cell activation, but the genome-wide roles of individual family members remain to be determined. c-Rel, a member of the NF-kappaB family, appears to play a specific role in T cell function because T cells from c-Rel(-/-) animals are defective in their response to immune signals. We have used expression profiling to identify sets of genes that are affected by either deletion or overexpression of c-Rel in T cells. Very few of these genes exhibit a strong requirement for c-Rel; rather, c-Rel appears to modulate the expression of a large number of genes in these cells. The sets of c-Rel-affected genes are significantly enriched for genes containing consensus NF-kappaB/Rel sites in their proximal promoter regions. In addition, their promoters contain a higher average density of NF-kappaB/Rel sites compared with all genes represented on the microarrays. A transcriptional module comprised of two closely spaced c-Rel consensus sites is found with higher frequency in the c-Rel-affected gene sets and may represent an important control module for genes regulated by c-Rel or other NF-kappaB family members. We confirmed the importance of these findings on a subgroup of genes by using quantitative PCR to monitor gene expression as well as in vitro c-Rel/DNA binding assays and luciferase reporter assays. The c-Rel-regulated genes identified here support a role for c-Rel in inflammatory responses as well as in the promotion of cell growth and survival.

Control of interleukin-2 gene transcription: a paradigm for inducible, tissue-specific gene expression.

Interleukin-2 (IL-2) is a key cytokine that controls immune cell function, in particular the adaptive arm of the immune system, through its ability to control the clonal expansion and homeostasis of peripheral T cells. IL-2 is produced almost exclusively by T cells in response to antigenic stimulation and thus provides an excellent example of a cell-specific inducible gene. The mechanisms that control IL-2 gene transcription have been studied in detail for the past 20 years and our current understanding of the nature of the inducible and tissue-specific controls will be discussed.

A role for Ets1, synergizing with AP-1 and GATA-3 in the regulation of IL-5 transcription in mouse Th2 lymphocytes.

IL-5 is a key regulator of eosinophilic inflammation and is selectively expressed by antigen-activated Th2 lymphocytes. An important role for the proximal AP-1 and GATA sites in regulating IL-5 transcription is generally accepted but the significance of an adjacent Ets/NFAT site has remained unclear. We have investigated its role using the mouse Th2 clone D10.G4.1. Transcription of IL-5 reporter gene plasmids could be induced in D10 cells by phorbol myristate acetate/cyclic adenosine monophosphate (PMA/cAMP) stimulation and significantly further enhanced by activation of the mitogen-activated protein (MAP) kinase pathways. Strong induction of IL-5 mRNA was also induced by PMA/cAMP. Mutagenesis showed that the Ets/NFAT site is of critical importance along with the AP-1 and GATA sites in regulating IL-5 transcription stimulated by PMA/cAMP and MAP kinase activation. Transactivation was used to investigate the transcription factors which could function at the three sites and possible synergistic interactions. AP-1 (c-Fos/c-Jun) strongly induced IL-5 transcription and dominant negative AP-1 constructs confirmed that AP-1 plays an important role in regulating IL-5 expression. Ets1, unlike other members of the Ets/NFAT family, synergized strongly with AP-1 suggesting that Ets1 is the family member which functions at the Ets/NFAT site. AP-1/Ets1 transactivation also stimulated IL-5 mRNA expression. Ets1 binding to the proximal promoter region, demonstrated by chromatin immunoprecipitation, was stimulated by PMA/cAMP. The absolute dependence on the binding sites for Ets1, AP-1 and GATA-3 together with the strong synergy between Ets1 and AP-1 suggest close cooperative interactions between the three transcription factors in the regulation of IL-5 expression in mouse T cells.