Carla J. Cohen

Endogenous retroviral LTRs as promoters for human genes: A critical assessment

Gene regulatory changes are thought to be major factors driving species evolution, with creation of new regulatory regions likely being instrumental in contributing to diversity among vertebrates. There is growing appreciation for the role of transposable elements (TEs) in gene regulation and, indeed, laboratory investigations have confirmed many specific examples of mammalian genes regulated by promoters donated by endogenous retroviruses (ERVs) or other TEs. Bioinformatics studies have revealed hundreds of additional instances where this is likely to be the case. Since the long terminal repeats (LTRs) of retroviruses naturally contain abundant transcriptional regulatory signals, roles for ERV LTRs in regulating mammalian genes are eminently plausible. Moreover, it seems reasonable that exaptation of an LTR regulatory module provides opportunities for evolution of new gene regulatory patterns. In this Review we summarize known examples of LTRs that function as human gene alternative promoters, as well as the evidence that LTR exaptation has resulted in a pattern of novel gene expression significantly different from the pattern before LTR insertion or from that of gene orthologs lacking the LTR. Available data suggest that, while new expression patterns can arise as a result of LTR usage, this situation is relatively rare and is largely restricted to the placenta. In many cases, the LTR appears to be a minor, alternative promoter with an expression pattern similar to that of the native promoter(s) and hence likely exerts a subtle overall effect on gene expression. We discuss these findings and offer evolutionary models to explain these trends.

Potential mechanisms of endogenous retroviral-mediated genomic instability in human cancer.

Malignancy results from a complex combination of genetic and epigenetic changes, the full effects of which are still largely unknown. Here we summarize current knowledge of the origin, retrotranspositional activity, epigenetic state, and transcription of human endogenous retroviruses (HERVs), and then discuss the potential effects of their deregulation in cancer. Evidence suggests that cancer-associated epigenetic changes most likely underlie potential HERV-mediated effects on genome and transcriptome instability and may play a role in malignancy. Despite our currently limited understanding of the importance of HERVs or other transposable elements in cancer development, we believe that the emerging era of high-throughput sequencing of cancer genomes, epigenomes, and transcriptomes will provide unprecedented opportunities to investigate these roles in the future.

Human Th1 and Th17 Cells Exhibit Epigenetic Stability at Signature Cytokine and Transcription Factor Loci

The linear model of Th cell lineage commitment is being revised due to reports that mature Th cells can trans-differentiate into alternate lineages. This ability of Th cells to reprogram is thought to be regulated by epigenetic mechanisms that control expression of transcription factors characteristic of opposing lineages. It is unclear, however, to what extent this new model of Th cell plasticity holds true in human Th cell subsets that develop under physiological conditions in vivo. We isolated in vivo-differentiated human Th1 and Th17 cells, as well as intermediate Th1/17 cells, and identified distinct epigenetic signatures at cytokine (IFNG and IL17A) and transcription factor (TBX21, RORC, and RORA) loci. We also examined the phenotypic and epigenetic stability of human Th17 cells exposed to Th1-polarizing conditions and found that although they could upregulate TBX21 and IFN-γ, this occurred without loss of IL-17 or RORC expression, and resulted in cells with a Th1/17 phenotype. Similarly, Th1 cells could upregulate IL-17 upon enforced expression of RORC2, but did not lose expression of IFN-γ or TBX21. Despite alterations in expression of these signature genes, epigenetic modifications were remarkably stable aside from the acquisition of active histone methylation marks at cytokine gene promoters. The limited capacity of human Th17 and Th1 cells to undergo complete lineage conversion suggests that the bipotent Th1/17 cells may arise from Th1 and/or Th17 cells. These data also question the broad applicability of the new model of Th cell lineage plasticity to in vivo-polarized human Th cell subsets.

Placenta-specific expression of the interleukin-2 (IL-2) receptor β subunit from an endogenous retroviral promoter.

Background: Long terminal repeats (LTRs) of endogenous retroviruses contain promoters active in cells. Results: An ancient human LTR is an alternative promoter for the IL2RB gene in placental trophoblast. Conclusion: The cytoplasmic signaling domain of IL2RB is present in placenta due to LTR activity. Significance: These findings implicate functions for IL2RB in the trophoblast and reveal an example of LTR exaptation to drive tissue-specific expression. The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression.

The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression

Objectives To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). Methods We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. Results The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10−14) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 ‘G’ allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk ‘A’ allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). Conclusion We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS.

An ankylosing spondylitis-associated genetic variant in the IL23R-IL12RB2 intergenic region modulates enhancer activity and is associated with increased Th1-cell differentiation.

Objectives To explore the functional basis for the association between ankylosing spondylitis (AS) and single-nucleotide polymorphisms (SNPs) in the IL23R-IL12RB2 intergenic region. Methods We performed conditional analysis on genetic association data and used epigenetic data on chromatin remodelling and transcription factor (TF) binding to identify the primary AS-associated IL23R-IL12RB2 intergenic SNP. Functional effects were tested in luciferase reporter assays in HEK293T cells and allele-specific TF binding was investigated by electrophoretic mobility gel shift assays. IL23R and IL12RB2 mRNA levels in CD4+ T cells were compared between cases homozygous for the AS-risk ‘A’ allele and the protective ‘G’ allele. The proportions of interleukin (IL)-17A+ and interferon (IFN)-γ+ CD4+ T-cells were measured by fluorescence-activated cell sorting and compared between these AS-risk and protective genotypes. Results Conditional analysis identified rs11209032 as the probable causal SNP within a 1.14 kb putative enhancer between IL23R and IL12RB2. Reduced luciferase activity was seen for the risk allele (p<0.001) and reduced H3K4me1 methylation observed in CD4+ T-cells from ‘A/A’ homozygotes (p=0.02). The binding of nuclear extract to the risk allele was decreased ∼3.5-fold compared with the protective allele (p<0.001). The proportion of IFN-γ+ CD4+ T-cells was increased in ‘A/A’ homozygotes (p=0.004), but neither IL23R nor IL12RB2 mRNA was affected. Conclusions The rs11209032 SNP downstream of IL23R forms part of an enhancer, allelic variation of which may influence Th1-cell numbers. Homozygosity for the risk ‘A’ allele is associated with more IFN-γ-secreting (Th1) cells. Further work is necessary to explain the mechanisms for these important observations.

SHIP-Deficient Dendritic Cells, Unlike Wild Type Dendritic Cells, Suppress T Cell Proliferation via a Nitric Oxide-Independent Mechanism

Background Dendritic cells (DCs) not only play a crucial role in activating immune cells but also suppressing them. We recently investigated SHIPs role in murine DCs in terms of immune cell activation and found that TLR agonist-stimulated SHIP−/− GM-CSF-derived DCs (GM-DCs) were far less capable than wild type (WT, SHIP+/+) GM-DCs at activating T cell proliferation. This was most likely because SHIP−/− GM-DCs could not up-regulate MHCII and/or co-stimulatory receptors following TLR stimulation. However, the role of SHIP in DC-induced T cell suppression was not investigated. Methodology/Principal Findings In this study we examined SHIPs role in DC-induced T cell suppression by co-culturing WT and SHIP−/− murine DCs, derived under different conditions or isolated from spleens, with αCD3+ αCD28 activated WT T cells and determined the relative suppressive abilities of the different DC subsets. We found that, in contrast to SHIP+/+ and −/− splenic or Flt3L-derived DCs, which do not suppress T cell proliferation in vitro, both SHIP+/+ and −/− GM-DCs were capable of potently suppressing T cell proliferation. However, WT GM-DC suppression appeared to be mediated, at least in part, by nitric oxide (NO) production while SHIP−/− GM-DCs expressed high levels of arginase 1 and did not produce NO. Following exhaustive studies to ascertain the mechanism of SHIP−/− DC-mediated suppression, we could conclude that cell-cell contact was required and the mechanism may be related to their relative immaturity, compared to SHIP+/+ GM-DCs. Conclusions These findings suggest that although both SHIP+/+ and −/− GM-DCs suppress T cell proliferation, the mechanism(s) employed are different. WT GM-DCs suppress, at least in part, via IFNγ-induced NO production while SHIP−/− GM-DCs do not produce NO and suppression can only be alleviated when contact is prevented.

Investigation of a possible extended risk haplotype in the IL23R region associated with ankylosing spondylitis

The IL23R region on chromosome 1 exhibits complex associations with ankylosing spondylitis (AS). We used publicly available epigenomic information and historical genetic association data to identify a putative regulatory element (PRE) in the intergenic region between IL23R and IL12RB2, which includes two single-nucleotide polymorphisms (SNPs) independently associated with AS—rs924080 (P=2 × 10−3) and rs11578380 (P=2 × 10−4). In luciferase reporter assays, this PRE showed silencer activity (P<0.001). Haplotype and conditional analysis of 4230 historical AS cases and 9700 controls revealed a possible AS-associated extended haplotype, including the PRE and risk variants at three SNPs (rs11209026, rs11209032 and rs924080), but excluding the rs11578380 risk variant. However, the rs924080 association was absent after conditioning on the primary association with rs11209032, which, in contrast, was robust to conditioning on all other AS-associated SNPs in this region (P<2 × 10−8). The role of this putative silencer on some IL23R extended haplotypes therefore remains unclear.

Evidence for a second ankylosing spondylitis-associated RUNX3 regulatory polymorphism

Objectives To explore the functions of RUNX3 single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS). Methods Individual SNP associations were evaluated in 4230 UK cases. Their effects on transcription factor (TF) binding, transcription regulation, chromatin modifications, gene expression and gene interactions were tested by database interrogation, luciferase reporter assays, electrophoretic mobility gel shifts, chromatin immunoprecipitation and real-time PCR. Results We confirmed the independent association of AS with rs4265380, which was robust (P=4.7×10−6) to conditioning on another nearby AS-associated RUNX3 SNP (rs4648889). A RUNX3 haplotype incorporating both SNPs was strongly associated with AS (OR 6.2; 95% CI 3.1 to 13.2, P=1.4×10−8). In a large UK cohort, rs4265380 is associated with leucocyte counts (including monocytes). RUNX3 expression is lower in AS peripheral blood mononuclear cells than healthy controls (P<0.002), independent of rs4265380 genotype. Enhancer function for this RUNX3 region was suggested by increased luciferase activity (approximately tenfold; P=0.005) for reporter constructs containing rs4265380. In monocytes, there was differential allelic binding of nuclear protein extracts to a 50 bp DNA probe containing rs4265380 that was strongly augmented by lipopolysaccharide activation. TF binding also included the histone modifier p300. There was enrichment for histone modifications associated with active enhancer elements (H3K27Ac and H3K79Me2) that may be allele dependent. Hi-C database interrogation showed chromosome interactions of RUNX3 bait with the nearby RP4-799D16.1 lincRNA. Conclusions The association of AS with this RUNX3 regulatory region involves at least two SNPs apparently operating in different cell types. Monocytes may be potential therapeutic targets in AS.

The severity of ankylosing spondylitis and responses to anti-tumour necrosis factor biologics are not influenced by the tumour necrosis factor receptor polymorphism incriminated in multiple sclerosis.

Genetic polymorphism (rs1800693) of TNFRSF1A (type 1 tumour necrosis factor receptor) encodes a potentially anti-inflammatory soluble truncated form of the p55 receptor, which is associated with predisposition to multiple sclerosis but protection against ankylosing spondylitis (AS). We analysed 2917 UK Caucasian cases by linear and logistic regression for associations of rs1800693 with disease severity assessed by the Bath Ankylosing Spondylitis measures of disease activity and function (BASDAI, BAS-G and BASFI) and/or responses to anti-TNF therapy. In contrast to predictions, rs1800693 GG homozygotes actually had significantly worse BASDAI (mean 4.2, 95% CI: 4–4.5) than AA homozygotes (mean 3.8, 95% CI: 3.7–4) in both the unadjusted (difference = 0.4, p = 0.006) and adjusted analyses (difference = 0.2–0.5, p = 0.002–0.04 depending on the adjustment model). We found no evidence that rs1900693 predicted functional status (BASFI) or global disease scores (BAS-G), and it exerted no influence on either the intention to treat with or efficacy of anti-TNF treatment.