RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Non-coding RNA regulation of integrins and their potential as therapeutic targets in cancer.

BACKGROUND: Integrins are integral to cell signalling and management of the extracellular matrix, and exquisite regulation of their expression is essential for a variety of cell signalling pathways, whilst disordered regulation is a key driver of tumour progression and metastasis. Most recently non-coding RNAs in the form of micro-RNA (miRNA) and long non-coding RNA (lncRNA) have emerged as a key mechanism by which tissue dependent gene expression is controlled. Whilst historically these molecules have been poorly understood, advances in 'omic' technologies and a greater understanding of non-coding regions of the genome have revealed that non-coding RNAs make up a large proportion of the transcriptome. CONCLUSIONS AND PERSPECTIVES: This review examines the regulation of integrin genes by ncRNAs, provides and overview of their mechanism of action and highlights how exploitation of these discoveries is informing the development of novel chemotherapeutic agents in the treatment of cancer. MiRNA molecules have been the most extensively characterised and negatively regulate most integrin genes, classically regulating genes through binding to recognition sequences in the mRNA 3'-untranslated regions of gene transcripts. LncRNA mechanisms of action are now being elucidated and appear to be more varied and complex, and may counter miRNA molecules, directly engage integrin mRNA transcripts, and guide or block both transcription factors and epigenetic machinery at integrin promoters or at other points in integrin regulation. Integrins as therapeutic targets are of enormous interest given their roles as oncogenes in a variety of tumours, and emerging therapeutics mimicking ncRNA mechanisms of action are already being trialled.

A novel long non-coding RNA regulates the integrin, ITGA2 in breast cancer.

PURPOSE: ITGA2 encodes the integrin, α2 which mediates metastatic progression, and is a predictor of poor prognosis and chemoresistance in breast cancer. Decreased ITGA2 promoter methylation is implicated as a driver of increased gene expression in aggressive prostate and pancreatic tumours, however the contribution of altered methylation to ITGA2 expression changes in breast tumours has not been examined. METHODS: ITGA2 gene methylation and gene expression was examined in publicly available breast cancer datasets, and ITGA2 promoter methylation was mapped by targeted bisulphite sequencing analysis in breast tumour cell lines. The expression of a putative regulatory long noncoding RNA (lncRNA) was examined by qPCR and its' functionality was investigated using gene knockdown (antisense oligonucleotides) and over expression in breast cancer cell lines. RESULTS: In breast tumours and breast cancer cell lines the ITGA2 promoter is largely unmethylated, with gene expression variable in tumour subtypes, irrespective of promoter methylation. A novel lncRNA (AC025180.1;ENSG00000249899), named herein I2ALR, was identified at the ITGA2 gene locus, and was variably expressed in breast tumours and breast cancer cell subtypes. I2LAR knockdown resulted in upregulation of ITGA2 gene expression, whilst over-expression of I2ALR resulted in downregulation of ITGA2 mRNA. Further, examination of two downstream targets of ITGA2 associated with breast tumor stemness and metastasis (CCND1 and ACLY), revealed concomitant gene expression changes in response to I2ALR modulation. CONCLUSION: I2ALR represents a novel regulatory molecule targeting ITGA2 expression in breast tumours; a finding of significant and topical interest to the development of therapeutics targeting this integrin.

Epigenetic regulation of the ITGB4 gene in prostate cancer.

BACKGROUND: Examination of epigenetic changes at the ITGB4 gene promoter reveals altered methylation at different stages of prostate tumour progression and these changes may, in part, explain the complex patterns of gene expression of this integrin observed. Transcriptional re-programming perturbs expression of cell adhesion molecules and underpins metastatic tumour cell behaviour. Decreasing expression of the cell adhesion molecule ITGB4, which encodes the beta subunit of the integrin, alpha6 beta4 (α6ß4), has been correlated with increased tumour aggressiveness and metastasis in multiple tumour types including prostate cancer. Paradoxically, in vitro studies in tumour cell models demonstrate that ITGB4 mediates cell mobility and invasion. Herein we examined whether transcriptional re-programming by methylation influenced ITGB4 gene expression at different stages of prostate cancer progression. Bisulphite sequencing of a large CpG island in the ITGB4 gene promoter identified differentially methylated regions in prostate cancer cell lines representing a localised tumour (22Rv1), lymph node metastasis (LNCaP), and a bone metastasis (PC-3). The highest levels of methylation were observed in the CpG island surrounding the ITGB4 transcription start site in PC-3 cells, and this observation also correlated with higher gene expression of ITGB4 in these cells. Furthermore, PC-3 cells expressed two distinct transcripts, using an alternate transcription start site, which was not detected in other cell lines. In prostate tumour biopsy samples, patterns of methylation across the ITGB4 promoter were similar overall in matched primary and metastatic samples (n = 4 pairs), with a trend toward loss of methylation at specific sites in metastatic lesions.

DNA methylation changes following DNA damage in prostate cancer cells.

Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.

Multiple endocrine neoplasia type 1: clinical correlates of MEN1 gene methylation.

Multiple endocrine neoplasia type 1 (MEN 1) has marked severity variation between individuals with the same mutation. To investigate any relationship between promoter methylation and clinical features, blood and tissue samples were collected from 16 members of the Tasman 1 MEN 1 kindred carrying a common splice site mutation and 7 patients with sporadic MEN 1. Methylation at 39 CpGs in the MEN1 promoter were assessed in formalin fixed, paraffin embedded parathyroid tissue. Clinical disease severity markers included age at first parathyroid operation, parathyroid hormone level and corrected serum calcium levels. Six patients with sporadic hyperparathyroidism were used for comparison. Minimal methylation was observed in all patients across CpG sites 1-23. In contrast, hypermethylation was observed at CpG sites 24-31 in MEN 1 patients, a pattern not observed in patients with non-MEN 1 parathyroid disease. Mean methylation at sites 24-31 was significantly correlated with age at first parathyroid operation (r = 0.652, p = 0.041). A permutation test, utilising the mean correlation coefficient (r = -0.401) revealed a possible association between relative PHPT severity and methylation score for each significant CpG site (p < 0.103). This novel study reveals evidence supporting a possible association between altered MEN1 promoter methylation and clinical severity of disease.

Distinct mechanisms of regulation of the ITGA6 and ITGB4 genes by RUNX1 in myeloid cells.

Integrins are transmembrane adhesion receptors that play an important role in hematopoiesis by facilitating interactions between hematopoietic cells and extracellular matrix components of the bone marrow and hematopoietic tissues. These interactions are important in regulating the function, proliferation, and differentiation of hematopoietic cells, as well as their homing and mobilization in the bone marrow. Not surprisingly altered expression and function of integrins plays a key role in the development and progression of cancer including leukemias. However, the regulation of integrin gene expression is not well characterized and the mechanisms by which integrin genes are disrupted in cancer remain unclear. Here we demonstrate for the first time that a key regulator of hematopoiesis, RUNX1, binds to and regulates the promoters of both the ITGA6 and ITGB4 genes in myeloid cells. The ITGA6 and ITGB4 integrin genes form the α6ß4 integrin receptor. However, our data indicate that RUNX1 functions differently at these two promoters. RUNX1 regulates ITGA6 through a consensus RUNX1 binding motif in its promoter. In contrast, although the ITGB4 promoter is also activated by RUNX1, it does so in the absence of a recognized consensus RUNX1 binding motif. Furthermore, our data suggest that regulation of ITGB4 may involve interactions between the promoter and upstream regulatory elements.

Comparison of pre-processing methodologies for Illumina 450k methylation array data in familial analyses.

BACKGROUND: Human methylome mapping in health and disease states has largely relied on Illumina Human Methylation 450k array (450k array) technology. Accompanying this has been the necessary evolution of analysis pipelines to facilitate data processing. The majority of these pipelines, however, cater for experimental designs where matched 'controls' or 'normal' samples are available. Experimental designs where no appropriate 'reference' exists remain challenging. Herein, we use data generated from our study of the inheritance of methylome profiles in families to evaluate the performance of eight normalisation pre-processing methods. Fifty individual samples representing four families were interrogated on five 450k array BeadChips. Eight normalisation methods were tested using qualitative and quantitative metrics, to assess efficacy and suitability. RESULTS: Stratified quantile normalisation combined with ComBat were consistently found to be the most appropriate when assessed using density, MDS and cluster plots. This was supported quantitatively by ANOVA on the first principal component where the effect of batch dropped from p < 0.01 to p = 0.97 after stratified QN and ComBat. Median absolute differences between replicated samples were the lowest after stratified QN and ComBat as were the standard error measures on known imprinted regions. Biological information was preserved after normalisation as indicated by the maintenance of a significant association between a known mQTL and methylation (p = 1.05e-05). CONCLUSIONS: A strategy combining stratified QN with ComBat is appropriate for use in the analyses when no reference sample is available but preservation of biological variation is paramount. There is great potential for use of 450k array data to further our understanding of the methylome in a variety of similar settings. Such advances will be reliant on the determination of appropriate methodologies for processing these data such as established here.

Fucoidan enhances the therapeutic potential of arsenic trioxide and all-trans retinoic acid in acute promyelocytic leukemia, in vitro and in vivo.

The morbidity and mortality associated with current therapies for acute promyelocytic leukemia (APL) remain a significant clinical concern, despite improvements in patient survival. Consequently, the development of adjuvant therapies that increase efficacy while reducing morbidities is important. Reducing the concentration of the toxic drugs in adjuvant therapy has the potential to reduce unwanted side effects. Therefore, this study aimed to determine the synergistic effects of fucoidan, an anti-tumor agent, with current APL therapies.When the human APL cell line, NB4, was treated in vitro with fucoidan plus ATO and ATRA at therapeutic and sub-therapeutic doses, there was an increase in sub-G0/G1 cells, annexin V/PI-positive-apoptotic cells and DNA fragmentation. This reduction in proliferation and increase in apoptosis was accompanied by enhanced myeloid differentiation as indicated by an increased expression of CD11b. This was not observed with the AML cell line Kasumi-1, suggesting specificity for APL.In vivo treatment of APL-bearing mice with fucoidan+ATRA or fucoidan+ATO delayed tumor growth, induced differentiation and increased tumor volume doubling time. The differentiated APL cells derived from the excised tumor mass exhibited decreased CD44 expression in fucoidan+ATRA treated mice. This could translate to decreased cell migration in APL patients.Our findings provide evidence supporting the use of fucoidan as an adjuvant therapeutic agent in the treatment of APL.

Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells In Vitro and In Vivo.

Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti-tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti-tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60, and K562 were treated with fucoidan and cell cycle, cell proliferation, and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase-dependent as it was significantly attenuated by pre-treatment with a pan-caspase inhibitor. P21/WAF1/CIP1 was significantly up-regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down-regulated the activation of AKT through hypo-phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia.

The Leukemia Inhibitory Factor Receptor Gene Is a Direct Target of RUNX1.

Activation of cytokine signaling via the leukemia inhibitory factor receptor (LIFR) plays an integral role in hematopoiesis, osteogenesis, and placental development, along with mediating neurotrophic mechanisms. However, the regulatory control of the LIFR gene has remained largely unexplored. Here, we characterize the LIFR gene as a novel target of the RUNX1 transcription factor. The RUNX1 transcription factor is an essential regulator of hematopoiesis and is a frequent target of point mutations and chromosomal alterations in leukemia. RUNX1 regulates hematopoiesis through its control of genes important for hematopoietic cell growth, proliferation, and differentiation, including a number of cytokines and cytokine receptors. LIFR is regulated by two alternate promoters: a placental-specific and a ubiquitously active general promoter. We show that both of these promoters are regulated by RUNX1. However, in myeloid cells LIFR expression is driven solely by the general LIFR promoter with our data indicating that the placental promoter is epigenetically silenced in these cells. While RUNX1 activates the LIFR general promoter, the oncogenic RUNX1-ETO fusion protein generated by the t(8;21) translocation commonly associated with acute myeloid leukemia represses promoter activity. The data presented here establish LIFR as a transcriptional target of RUNX1 and suggest that disruption of RUNX1 activity in myeloid cells may result in altered LIFR signaling in these cells.

Interplay between Transcription Factors and the Epigenome: Insight from the Role of RUNX1 in Leukemia.

The genome has the ability to respond in a precise and co-ordinated manner to cellular signals. It achieves this through the concerted actions of transcription factors and the chromatin platform, which are targets of the signaling pathways. Our understanding of the molecular mechanisms through which transcription factors and the chromatin landscape each control gene activity has expanded dramatically over recent years, and attention has now turned to understanding the complex, multifaceted interplay between these regulatory layers in normal and disease states. It has become apparent that transcription factors as well as the components and modifiers of the epigenetic machinery are frequent targets of genomic alterations in cancer cells. Through the study of these factors, we can gain unique insight into the dynamic interplay between transcription factors and the epigenome, and how their dysregulation leads to aberrant gene expression programs in cancer. Here, we will highlight how these factors normally co-operate to establish and maintain the transcriptional and epigenetic landscape of cells, and how this is reprogramed in cancer, focusing on the RUNX1 transcription factor and oncogenic derivative RUNX1-ETO in leukemia as paradigms of transcriptional and epigenetic reprograming.

Fucoidan and cancer: a multifunctional molecule with anti-tumor potential.

There is a wide variety of cancer types yet, all share some common cellular and molecular behaviors. Most of the chemotherapeutic agents used in cancer treatment are designed to target common deregulated mechanisms within cancer cells. Many healthy tissues are also affected by the cytotoxic effects of these chemical agents. Fucoidan, a natural component of brown seaweed, has anti-cancer activity against various cancer types by targeting key apoptotic molecules. It also has beneficial effects as it can protect against toxicity associated with chemotherapeutic agents and radiation. Thus the synergistic effect of fucoidan with current anti-cancer agents is of considerable interest. This review discusses the mechanisms by which fucoidan retards tumor development, eradicates tumor cells and synergizes with anti-cancer chemotherapeutic agents. Challenges to the development of fucoidan as an anti-cancer agent will also be discussed.

Genetic Determinants of Epigenetic Patterns: Providing Insight into Disease.

The field of epigenetics and our understanding of the mechanisms that regulate the establishment, maintenance and heritability of epigenetic patterns continue to grow at a remarkable rate. This information is providing increased understanding of the role of epigenetic changes in disease, insight into the underlying causes of these epigenetic changes and revealing new avenues for therapeutic intervention. Epigenetic modifiers are increasingly being pursued as therapeutic targets in a range of diseases, with a number of agents targeting epigenetic modifications already proving effective in diseases such as cancer. Although it is well established that DNA mutations and aberrant expression of epigenetic modifiers play a key role in disease, attention is now turning to the interplay between genetic and epigenetic factors in complex disease etiology. The role of genetic variability in determining epigenetic profiles, which can then be modified by environmental and stochastic factors, is becoming more apparent. Understanding the interplay between genetic and epigenetic factors is likely to aid in identifying individuals most likely to benefit from epigenetic therapies. This goal is coming closer to realization because of continual advances in laboratory and statistical tools enabling improvements in the integration of genomic, epigenomic and phenotypic data.

Regulation of the ITGA2 gene by epigenetic mechanisms in prostate cancer.

BACKGROUND: Integrin alpha2 beta1 (α2 ß1 ) plays an integral role in tumour cell invasion, metastasis and angiogenesis, and altered expression of the receptor has been linked to tumour prognosis in several solid tumours. However, the relationship is complex, with both increased and decreased expression associated with different stages of tumour metastases in several tumour types. The ITGA2 gene, which codes for the α2 subunit, was examined to investigate whether a large CpG island associated with its promoter region is involved in the differential expression of ITGA2 observed in prostate cancer. METHODS: Bisulphite sequencing of the ITGA2 promoter was used to assess methylation in formalin-fixed paraffin-embedded (FFPE) prostate tumour specimens and prostate cancer cell lines, PC3, 22Rv1 and LNCaP. Changes in ITGA2 mRNA expression were measured using quantitative PCR. ITGA2 functionality was interrogated using cell migration scratch assays and siRNA knockdown experiments. RESULTS: Bisulphite sequencing revealed strikingly decreased methylation at key CpG sites within the promoter of tumour samples, when compared with normal prostate tissue. Altered methylation of this CpG island is also associated with differences in expression in the non-invasive LNCaP, and the highly metastatic PC3 and 22Rv1 prostate cancer cell lines. Further bisulphite sequencing confirmed that selected CpGs were highly methylated in LNCaP cells, whilst only low levels of methylation were observed in PC3 and 22Rv1 cells, correlating with ITGA2 transcript levels. Examination of the increased expression of ITGA2 was shown to influence migratory potential via scratch assay in PC3, 22Rv1 and LNCaP cells, and was confirmed by siRNA knockdown experiments. CONCLUSIONS: Taken together, our data supports the assertion that epigenetic modification of the ITGA2 promoter is a mechanism by which ITGA2 expression is regulated.

RNA-seq profiling of a radiation resistant and radiation sensitive prostate cancer cell line highlights opposing regulation of DNA repair and targets for radiosensitization.

BACKGROUND: Radiotherapy is a chosen treatment option for prostate cancer patients and while some tumours respond well, up to 50% of patients may experience tumour recurrence. Identification of functionally relevant predictive biomarkers for radioresponse in prostate cancer would enable radioresistant patients to be directed to more appropriate treatment options, avoiding the side-effects of radiotherapy. METHODS: Using an in vitro model to screen for novel biomarkers of radioresistance, transcriptome analysis of a radioresistant (PC-3) and radiosensitive (LNCaP) prostate cancer cell line was performed. Following pathway analysis candidate genes were validated using qRT-PCR. The DNA repair pathway in radioresistant PC-3 cells was then targeted for radiation sensitization using the PARP inhibitor, niacinimide. RESULTS: Opposing regulation of a DNA repair and replication pathway was observed between PC-3 and LNCaP cells from RNA-seq analysis. Candidate genes BRCA1, RAD51, FANCG, MCM7, CDC6 and ORC1 were identified as being significantly differentially regulated post-irradiation. qRT-PCR validation confirmed BRCA1, RAD51 and FANCG as being significantly differentially regulated at 24 hours post radiotherapy (p-value =0.003, 0.045 and 0.003 respectively). While the radiosensitive LNCaP cells down-regulated BRCA1, FANCG and RAD51, the radioresistant PC-3 cell line up-regulated these candidates to promote cell survival post-radiotherapy and a similar trend was observed for MCM7, CDC6 and ORC1. Inhibition of DNA repair using niacinamide sensitised the radioresistant cells to irradiation, reducing cell survival at 2 Gy from 66% to 44.3% (p-value =0.02). CONCLUSIONS: These findings suggest that the DNA repair candidates identified via RNA-seq hold potential as both targets for radiation sensitization and predictive biomarkers in prostate cancer.

The potential role of epigenetic modifications in the heritability of multiple sclerosis.

It is now well established that both genetic and environmental factors contribute to and interact in the development of multiple sclerosis (MS). However, the currently described causal genetic variants do not explain the majority of the heritability of MS, resulting in 'missing heritability'. Epigenetic mechanisms, which principally include DNA methylation, histone modifications and microRNA-mediated post-transcriptional gene silencing, may contribute a significant component of this missing heritability. As the development of MS is a dynamic process potentially starting with inflammation, then demyelination, remyelination and neurodegeneration, we have reviewed the dynamic epigenetic changes in these aspects of MS pathogenesis and describe how environmental risk factors may interact with epigenetic changes to manifest in disease.

Epigenetic regulation of inducible gene expression in the immune system.

T cells are exquisitely poised to respond rapidly to pathogens and have proved an instructive model for exploring the regulation of inducible genes. Individual genes respond to antigenic stimulation in different ways, and it has become clear that the interplay between transcription factors and the chromatin platform of individual genes governs these responses. Our understanding of the complexity of the chromatin platform and the epigenetic mechanisms that contribute to transcriptional control has expanded dramatically in recent years. These mechanisms include the presence/absence of histone modification marks, which form an epigenetic signature to mark active or inactive genes. These signatures are dynamically added or removed by epigenetic enzymes, comprising an array of histone-modifying enzymes, including the more recently recognized chromatin-associated signalling kinases. In addition, chromatin-remodelling complexes physically alter the chromatin structure to regulate chromatin accessibility to transcriptional regulatory factors. The advent of genome-wide technologies has enabled characterization of the chromatin landscape of T cells in terms of histone occupancy, histone modification patterns and transcription factor association with specific genomic regulatory regions, generating a picture of the T-cell epigenome. Here, we discuss the multi-layered regulation of inducible gene expression in the immune system, focusing on the interplay between transcription factors, and the T-cell epigenome, including the role played by chromatin remodellers and epigenetic enzymes. We will also use IL2, a key inducible cytokine gene in T cells, as an example of how the different layers of epigenetic mechanisms regulate immune responsive genes during T-cell activation.

Depletion of c-Rel from cytokine gene promoters is required for chromatin reassembly and termination of gene responses to T cell activation.

The role of the Nuclear Factor κB (NF-κB) transcription factor family in T cell function has been well described. The c-Rel family member is of particular importance in initiating T cell responses to antigen and regulating activation of inflammatory cytokine genes, including the Interleukin-2 (IL-2) and Granulocyte macrophage colony stimulating factor (GM-CSF) genes. c-Rel is required for chromatin remodeling of these gene promoters, which involves depletion of histones from the promoters in response to T cell activating signals. These chromatin remodeling events precede transcriptional activation of the genes. The subsequent down-regulation of cytokine gene expression is important in the termination of an immune response and here we examine this process at the murine GM-CSF and IL-2 genes. We show that the cytokine mRNA levels rapidly return to basal levels following stimulus removal and this is associated with reassembly of histones onto the promoter. Histone reassembly at the GM-CSF and IL-2 promoters occurs concomitantly with depletion of RelA, c-Rel and RNA polymerase II from the promoters. Furthermore we show that transcriptional down-regulation and chromatin reassembly is dependent on depletion of c-Rel from the nucleus, and that this is regulated by the nuclear translocation of the NF-κB inhibitor, IκBα. The nuclear activation of c-Rel therefore not only regulates the initiation of GM-CSF and IL-2 gene activation in response to T cell activation, but also the termination of these gene responses following the removal of the activating signal.

Olfactory ensheathing cells moderate nuclear factor kappaB translocation in astrocytes.

Nuclear factor kappaB (NFκB) is a key transcriptional regulator of inflammatory genes. We investigated the modulatory effects of olfactory ensheathing cells (OECs), microglia and meningeal fibroblasts on translocation of NFκB to astrocyte nuclei. The percentage of activated astrocytes in co-cultures with OECs was significantly less than for co-cultures with microglia (p<0.001) and fibroblasts (p<0.05). Phorbol myristate acetate (PMA) and calcium ionophore stimulation of p65 NFκB translocation to nuclei provided an in vitro model of astrocyte inflammatory activation. Soluble factors released by OECs significantly moderated the astrocytic NFκB translocation induced by either PMA/calcium ionophore or microglia-derived factors (p<0.001). Insulin-like growth factor-1 may contribute to these effects, since it is expressed by OECs and also significantly moderated the astrocytic NFκB translocation (p<0.05), albeit insufficiently to fully account for the OEC-induced moderation (p<0.01). Olfactory ensheathing cells significantly moderated the increased transcription of the pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor in the activated astrocytes (p<0.01). These results suggest that transplanted OECs could improve neural repair after CNS injury by moderating astrocyte activation.